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Phytochemical analysis and pharmacological evaluation of methanolic leaf extract of Moringa oleifera Lam. in ovalbumin induced allergic asthma
South African Journal of Botany 130 (2020) 484 493
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Phytochemical analysis and pharmacological evaluation of methanolic leaf extract of Moringa oleifera Lam. in ovalbumin induced allergic asthma Sandhya Suresha,b,1, Abu Sufiyan Chhipaa,b,1,*, Mansi Guptaa,b, Sunali Lalotraa,b, S.S. Sisodiab, Ruma Baksia,c, Manish Nivsarkara a
Department of Pharmacology and Toxicolcogy, B.V. Patel PERD Centre, Ahmedabad, Gujarat, India Faculty of Pharmacy, Bhupal Noble’s University, Udaipur, Rajasthan, India c Registered PhD scholar (External) at Faculty of Science, NIRMA University, Sarkhej-Gandhinagar Highway, Ahmedabad, Gujarat 382481, India b
A R T I C L E
I N F O
Article History: Received 16 July 2019 Revised 25 December 2019 Accepted 30 January 2020 Available online xxx Edited by AR Ndhlala Keywords: Asthma Moringa oleifera MOLE Bronchoconstriction Histamine
A B S T R A C T
Aim of the study: The present study was carried out to evaluate the anti-asthmatic effects of Moringa oleifera Lam. leaf extract (MOLE) on ovalbumin-induced asthma in guinea pigs. Materials and method: Extraction, preliminary phytochemical screening and HPTLC fingerprinting analysis of MOLE was carried out. Total phenolics and flavonoids in the extract were determined. Evaluation of antiasthmatic effect of the extract was done in guinea pigs sensitized with OVA albumin. Dexamathasone was taken as standard drug, while the test extract was administered at two dose levels before challenging the experimental animals with aerosolized 0.5% OVA albumin. Animals were randomized and divided into 5 groups namely normal control (NC), disease control (DC), positive control (PC), Test group 1 (T-1) and Test group 2 (T-2). Bronchoconstriction and lung function tests were performed. At the end of study, blood was collected from animals in each group to take total WBC count. Leucocyte count was also taken from Bronchoalveolar lavage fluid (BAL) collected from the lungs of animals belonging to each group. Histamine estimation in lung tissues was performed. Changes in lung tissue were observed by histopathological study. Results: Extractive value of MOLE was 24.9%. Phytochemical screening of extract suggested that the extract is rich in broad classes of phytoconstituents including flavonoids, phenolics, glycosides and tannins. Fingerprint analysis was conclusive of the presence of multiple compounds in the extract. Total phenolics in the extract were found to be 9.8 g equivalent of gallic acid while the flavonoidal content was 0.46 g equivalent of Catechin. Administration of standard and test extracts resulted in improved lung functions. Conclusion: Methanolic extract of Moringa oleifera leaf has beneficial effects against bronchoconstriction, airway inflammation and asthma. Further exploratory studies may lead to identification and isolation of potential anti-asthmatic candidate molecules from the plant. © 2020 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Asthma is a chronic inflammatory disease characterized by increased responsiveness of trachea and bronchi to multiple stimuli. This allergic disease manifests as airway obstructions that involves inflammation of the pulmonary airways and bronchial hyper responsiveness (Education et al., 2007; Shifren et al., 2012). Various inflammatory cells come into play during asthma and bronchoconstriction. These include eosinophils, lymphocytes, neutrophils monocytes, histamine, cytokines, leukotriene, prostaglandins and thromboxanes (Taur and Patil, 2011b; Pedersen et al., 1996; Andrade and de Sousa,
* Corresponding author at: 17, Chhipa Bakhal, Khanpura, Mandsaur, Madhya Pradesh 458001, India. E-mail address: asufi[email protected] (A.S. Chhipa). 1 Authors contributed equally. https://doi.org/10.1016/j.sajb.2020.01.046 0254-6299/© 2020 SAAB. Published by Elsevier B.V. All rights reserved.
2013; Kapoor et al., 2011; Wang et al., 2016; Machado-Carvalho et al., 2014; Arimura et al., 1992; Okechukwu and Ekeuku, 2012). The associated side effects of currently available medications for the treatment of asthma makes it imperative for the researchers to search for more effective drugs with least or no side effects. Herbal medicines are promising alternatives to these interventions owing to their acceptability among patients, efficacy and least side effects (Patel et al., 2020). Medicinal plants with significant anti-inflammatory, immunomodulatory, antihistaminic and smooth-muscle relaxant activity are proved to be beneficial in the management of asthma (Taur and Patil, 2011a). Natural products from plants sources have been utilized for the alleviation of human illness since the ancient times (Chhipa and Sisodia, 2019). Isolation and identification of flavonoids, glycosides, tannins, alkaloids, saponins and other phytochemicals from plant origin have been useful in controlling various inflammatory and allergic
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responses (Lee et al., 2011; Kimura et al., 1987; Khan, 2015; Zdanowicz, 2007; Sung et al., 2017). Flavonoids such as quercetin, rutin, apigenin and kaempferol are known contributors in the prevention of airway inflammatory responses and asthma (Li et al., 2010; Jung et al., 2007; Wang et al., 2018). Moringa oleifera Lam. is a drought resistant tree that belongs to family Moringaceae (Kou et al., 2018). It is widely utilized traditionally for its effectiveness against wide range of diseases and disorders including hyperglycaemia, lithiasis, hypertension, microbial, fungal viral infections, hepatotoxicity, inflammation and fever. Importantly, the plant parts are also utilized in the treatment of asthma (Rao et al., 1999; Denny Fitriana et al., 2016; Ingale and Gandhi, 2016; El-bakry et al., 2016; Khan et al., 2017; Mishra et al., 2011). The plant also has recognition in ayurvedic and unani systems of medicine (Mughal et al., 1999). Different parts of the plant are found to be rich in a plethora of phytoconstituents. These include alkaloids, flavonoids, tannins, quinine, saponins, glycosides and fixed oils (Paikra et al., 2017). However, these phytoconstituents are found to be nonuniformly present in different parts of the plant. For instance, flavonoidal glycosides (quercetin, kaempferol and isorhamnetin) are present mostly in leaves while the roots and seeds are devoid of flavonoidal contents (Saini et al., 2016; Aja et al., 2014; Vergara-Jimenez et al., 2017; Makita et al., 2016; Sulastri et al., 2018b; Lin et al., 2018). Moreover, previous studies on phytochemical screening of the seeds and leaves of the plant suggest that the leaves carry higher phytochemical composition in comparison to the seeds of Moringa Oleifera (Aja et al., 2014; Vergara-Jimenez et al., 2017). Preclinical studies have showed significant anti-inflammatory, anaphylactic and antiasthmatic effects of the seeds of Moringa oleifera (Mahajan et al., 2007; Agrawal and Mehta, 2008; Mahajan and Mehta, 2008b). However, the leaves of the plant are less studied in terms of their anti-inflammatory and anti-asthmatic effects. Moreover, based on the phytochemical composition of leaves which is much higher than the seeds of the plant (Aja et al., 2014; Vergara-Jimenez et al., 2017), higher efficiency of leaves against inflammation and asthmatic responses is undoubtedly perceivable. Based on these findings, we have attempted to evaluate the anti-asthmatic effects of methanolic extract of Moringa oleifera leaves in ova-albumin induced airway inflammation and asthma in guinea pigs. The extract was tested at two dose levels (250 mg/kg and 500 mg/kg). Dexamethasone is an effective anti-asthmatic agent that is used to treat asthma (Cross et al., 2011; Shefrin and Goldman, 2009). It is a corticosteroid drug that exerts its action by suppressing the late phase inflammation and chemotaxis in asthma (Townley and Suliaman, 1987). Based on the previous reports of the plant as an anti-inflammatory and antianaphylactic agent, we have used dexamethasone as the standard drug to compare the efficacy of the leaf extract with standard interventions in controlling airway inflammation and asthmatic responses. Moreover, to evaluate the effects of the extract in the alleviation of human allergic asthma, guinea pigs sensitized with ovalbumin allergen is a preferred animal model. In that view, the present study was conducted in ovalbumin sensitized guinea pigs to explore the potential of methanolic extract of leaves of Moringa oleifera in controlling airway inflammation and asthma. 2. Materials and methods 2.1. Reagents Methanol, Dragondroff’s reagent, chloroform, Sodium hydroxide, sodium sulphate, Ferric chloride, copper sulphate, toluene, ethyl acetate, Folin Coicalteu reagent, sodium carbonate, Gallic acid, Sodium nitroxide, Aluminium trichloride, perchloric acid, hydrochloric acid were obtained from the department of Pharmacology and Toxicology, B.V. Patel PERD center. Dexamethasone, acetylcholine and histamine were purchased from MP biomedicals. Aluminum hydroxide, catechin, Agar, Formaldehyde and Sodium chloride were purchased from
485
fisher scientific. n-Butanol and n-Heptane were purchased from Merck specialities Pvt. Ltd. Isoflurane and Ovalbumin were purchased from Raman and well Pvt. Ltd. and HIMEDIA respectively. All chemicals and reagents were of analytical grade. 2.2. Procurement and identification of the plant material Leaves of Moringa oleifera Lam. were collected from the medicinal plant garden of B.V. Patel PERD center, Ahmedabad in the month of October 2018. Plant material was identified and authenticated by taxonomist in Department of Pharmacognosy and Phytochemistry, B.V. PERD center. A voucher specimen was submitted in the department for future reference. Voucher specimen number: BVPPERD/PP/1118/08. 2.3. Extraction of plant material Moringa oleifera leaves were shade dried for 1 week at 37°C. Dried leaves were powdered and the weight of coarse material was taken. For extraction, 350 g material was taken in 1litre of methanol in the conical flasks in two sets such that each flask receives 500 ml of methanol. Flasks were kept overnight. After 3 days the solutions in the flasks were filtered and kept on a rotary evaporator at 50°C to evaporate the solvent. The process was repeated 3 times with the remaining marc. The extract obtained after evaporation was combined and percent yield of Moringa oleifera leaf extract (MOLE) was calculated (Vongsak et al., 2013; Mahdi et al., 2016). 2.4. Preliminary phytochemical screening of extract Preliminary phytochemical analysis of extract was performed for the identification of broad classes of phytoconstituents including alkaloid, steroid, flavonoid, phenolics, glycosides, tannins and saponins according to their standard method (Ezeonu and Ejikeme, 2016; Dahiru et al., 2006). 2.5. Phytochemical fingerprinting analysis High performance Thin Layer Chromatography (HPTLC) was done for fingerprinting analysis of the extract using Hexane: Ethyl acetate: Acetic acid as the solvent system taken in the ratio 5:4:1. Solvent system was prepared (10 ml) and kept for 20 min in the TLC chamber for saturation to take place. For HPTLC analysis, the test solution (15 ng) was applied in the form of band (8 mm) by using 100 mL of CAMAG syringe on 10 £ 10 cm aluminium packed TLC plate with the help of a 100mL Hamilton syringe and Linomat V applicator attached with HPTLC system programmed through Win CATS software. TLC plates were developed by the ascending technique using 10 ml of mobile phase [Hexane: Ethyl acetate: Acetic acid] taken in the ratio 5:4:1 in a CAMAG twin-through glass chamber and saturated with mobile phase covered with a stainless-steel lid. TLC plate was dried. The image of the plate was taken in daylight and then in photo-documentation chamber. Images were captured at 254 nm, 366 nm and 530 nm (after derivatisation with Anisaldehyde Sulphuric acid reagent). Densitometric scanning was performed with scanner equipped with Win CATS package at λmax = 254, 366 and 530 nm. Peak tables, Rf values, and HPTLC chromatograms were recorded (Swathi, 2016; Elangovan et al., 2015). 2.6. Estimation of total phenolic contents by spectrophotometry Powdered material (Moringa oleifera leaf) of the plant was weighed and extracted with 10 ml of 50% aqueous methanol and the solution was filtered and collected. The final volume was made up with 50 ml methanol. From this, 0.1 ml solution was taken and added in 10 ml of double distilled water and 1.5 ml of Folin-Ciocalteu reagent in a 25 ml volumetric flask followed by incubation for 5 min.
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4 ml of 20% sodium carbonate was added and the final volume was made to 25 ml with double distilled water. Solution was mixed thoroughly and incubated at room temperature for 30 min. The absorption was recorded at 765 nm. Gallic acid (100 mg/ml) was used as the standard phenolic (Sulastri et al., 2018a; Tabasum et al., 2016). 2.7. Estimation of total flavonoids contents by photometry For the estimation of total flavonoids in the extract, 125mL of extract solution in methanol was taken and 75 mL of 5% NaNO2 solution was added in it. The mixture was allowed to stand for 6 min. followed by addition of 150mL of aluminium trichloride (10%) and incubation for 5 min. Thereafter, 750mL of NaOH (1 M) was added to the mixture and final volume of the solution was adjusted to 2.5 ml with distilled water. The mixture was allowed to turn pink and final the absorbance was measured at 510 nm. Catechin was taken as standard flavonoid and total flavonoids content was expressed as gram et al., 2018). equivalent of catechin (Okumu et al., 2016; Nobosse 2.8. Animal husbandry and feeds Dunkin-Hartley guinea pigs (350 450 g) of either sex were housed in a room maintained at 25 °C with a relative humidity of 65% in 14-hr light-dark cycle. Nutritional solutions and green grass were given ad libitum to the animals and filtered tap water was kept for drinking. All experiments were performed under ethical guidelines. The protocol was reviewed and approved by the Institutional animal ethics committee (IAEC), Protocol no: PERD/IAEC/2018/015. Through the entire experimental period, growth, health, and food intake capacity of animals were monitored and maintained. 2.9. Sensitization and treatment of animals Animals were randomised in five different groups (n = 6/group). Group I: normal control (NC) receiving agar solution, Group II disease control (DC) (Ovalbumin sensitized), Group III positive control (PC) receiving dexamethasone (Ovalbumin sensitized then received 2.5 mg/kg dexamethasone according to their body weights), Group IV test control (T-1) receiving Moringa oleifera leaf extract (Ovalbumin sensitized then received 250 mg/kg MOLE according to body weight), group V test control (T-2) receiving Moringa oleifera leaf extract (Ovalbumin sensitized then received 500 mg/kg MOLE according to body weight. All selected animals (except group I) were sensitized with 100 mg of ovalbumin absorbed into aluminium hydroxide saline solution by using a subcutaneous injection on day 0 as the first sensitization. Animals were sensitized for the second time on day 14. Dosing of the test drug and positive control are started from day 14 to 21, one hour prior to sensitization (Mahajan and Mehta, 2008a). 2.10. Ovalbumin exposure On day 18 to day 21, animals in each group (except group I) were challenged with aerosolized 0.5% OVA for 2 min., 2.5 h after receiving their drug or vehicle treatment. For the challenge, conscious animals were placed into a plastic circular chamber (diameter = 70 cm, height = 40 cm) connected to a nebulizer (CX4-Omron Healthcare Company Ltd. Kyoto, Japan). Animals in the group I were exposed to aerosolized saline (Mahajan and Mehta, 2008a; Meurs et al., 2006). 2.11. Haematology On day 17 and 22, blood from animals in each group was collected and total leucocyte count was measured in an automated haematology analyser (VetScan HM-5; Abaxis Inc., Union City, CA, USA) (31).
2.12. Measurement of lung function test On day 21, animals from each group were subjected to lung function parameter measurements. Tidal volume and respiratory rate in guinea pigs were measured before and after the aerosolized ovalbumin exposure with the help of nebulizer (OMRON, NE C-25 model) and recordings were taken in the biopac system (model MP-35, Biopac System, Inc., Santa Barbara, CA). Animals in group I (normal control) animals were exposed to aerosolized saline (Mahajan and Mehta, 2008a). 2.13. Measurement of bronchoconstriction test After the measurement of the lung function parameters, all animals were subjected to a bronchoconstriction test. OVA-sensitized hosts were exposed (in a conscious state) to 0.25% acetylcholine (ACH) solution for 30 s using a nebulizer (OMRON, NE C-25 model.) connected to the animal holder. Guinea pigs in the normal control group were exposed to normal saline in place of Ach. In each case, before and after the exposure of Ach, the tidal volume and respiration rate of each animal was noted (Mahajan and Mehta, 2008a). 2.14. Collection of bronchoalveolar lavage (BAL) fluid For the collection of BAL fluid, guinea pigs were anesthetized by isoflurane in the anaesthesia chamber. A polypropylene cannula was inserted into the trachea and 0.9% (w/v) normal saline solution (10 ml) was introduced into the lungs using a 10-ml syringe. The injected saline was recovered 5 min later. The recovered lavage fluid (5 ml) was centrifuged at 5000 x g for 10 min at 4 °C; the resultant supernatant was discarded and the cells in the pellet were washed in 0.5 ml saline and total cell counts were then performed in Automated haematology analyser (VetScan HM-5; Abaxis Inc., Union City, CA, USA) (Mahajan and Mehta, 2008a). 2.15. Histamine assay in lung tissue Lung tissues from animals belonging to different groups were collected and used for the estimation of histamine. Around 200 g of lung tissue was mixed with 2.5 ml normal saline and 2.5 ml of 0.8 N perchloric acid followed by centrifugation (4000 £ g, 7 min at 4 °C). The resulting supernatant (2 ml) was taken in a test tube containing 0.25 ml of 5 N NaOH, 0.75 g NaCl, and 5 ml n-butanol. The mixture was then was vortexed for 5 min to ensure the histamine localization into the butanol followed by centrifugation. The aqueous phase after centrifugation was discarded while the organic phase was washed with 2.5 ml salt-saturated 0.1 N NaOH solution to ensure the removal of any residual histidine. Thereafter, the mixture was centrifuged again and butanol was taken to a test tube containing 2 ml of 0.1 N HCl and 5 ml n-heptane. The mixture was again centrifuged and the presence of histamine determined fluorometrically (Shore et al., 1959; Jannatin et al., 2017). 2.16. Histopathological analysis Histopathology analysis of lung tissues obtained from each guinea pig was carried out to study the effect of extract on the immunemediated chronic inflammation brought about by sensitization with OVA. Dissected lungs and trachea were washed with normal saline and then placed in a 10% formaldehyde solution for 1week. After the tissues were fixed, specimens of lung tissues were embedded in paraffin wax and 5pm sections were cut and stained with haematoxylin and eosin dye for morphology. Images of selected sections were captured at 40X magnifications using a zoom digital camera optical microscopy (IX 51; Olympus, Tokyo, Japan) equipped with a digital camera (TL4) (Mahajan and Mehta, 2008a).
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2.17. Statistical analysis
3.5. Effect of MOLE on body weight
Results were reported as mean § SEM. Statistical analysis was performed using one-way analysis of variance ANOVA and t-test using graph pad prism 8 software. The data was considered statistically significant at p < 0.05.
All animals in diseased and treatment groups (Positive and test control) did not show any significant difference in body weight during experimental period in comparison to those present in normal control group. The result is suggestive that MOLE did not interfere with normal growth of animals (Fig. 5).
3. Results 3.1. Extraction and preliminary phytochemical study The percent yield of methanolic extract of Moringa oleifera leaf (MOLE) was found to be 24.9%. The preliminary phytochemical screening of MOLE showed the presence of flavonoids, glycosides, tannins, and phenolics.
3.6. Effect of MOLE on leucocyte count in the blood WBC counts were significantly different among different groups. The administration of standard drug OVA+DEX and extract at both dose levels (MOLE 250 mg/kg and MOLE 500 mg/kg) lowered the leucocyte count significantly as compared to disease group (*p < 0.05) on days 17 and 22. Reduction in WBC count was maximum in positive control group receiving standard drug (OVA+DXM) followed by test groups receiving MOLE (250 mg/kg and 500 mg/kg) (Fig. 6).
3.2. HPTLC fingerprinting analysis Peaks corresponding to various phytoconstituents were obtained. TLC plate under CAMAG HPTLC system resulted in three different chromatograms at 254 nm, 366 nm and 530 nm at 7, 10 and 17 peaks respectively (Figs. 1 and 2). Ranges of Rf values were in between 0.10 0.98, 0.13 0.96 and 0.05 0.98 under these wavelength (Table 1).
3.7. Effect of MOLE on leucocyte count in BAL fluid All groups receiving interventions (Positive control and test groups) showed significant reduction in WBC in comparison to disease group (p<0.05) (Fig. 7). However, maximum reduction in WBC counts was observed in positive control (OVA+DXM) followed by test groups receiving higher dose (500 mg/kg) and lower dose (250 mg/ kg) of MOLE.
3.3. Spectrophotometric estimation of total phenolic contents 3.8. Effect of MOLE on histamine level Total phenolics in the extract were calculated from the standard curve of gallic acid (Fig. 3). Concentration of total phenolic was found to be 9.8 mg/ ml equivalent of Gallic acid (Table 2).
3.4. Spectrophotometric estimation of total flavonoid Flavonoid contents in the extract were obtained from the standard curve of Catechin (Fig. 4). Total flavonoids in the extract were found to be 0.46 mg/ml (Table 3).
Similar results were obtained in case of histamine levels in different groups. Maximum reduction in histamine levels was noticed in positive control group (OVA+DEX) (Fig. 8). Test groups receiving the MOLE at both dose levels (250 mg/kg and 500 mg/kg) also showed significant reduction in histamine levels in comparison to diseased group (*p < 0.05) (Table 4). 3.9. Effects on respiratory rate (Ovalbumin exposure) Respiratory rate was less in groups receiving standard (OVA + DEX) and test drugs (OVA + MOLE) (Fig. 9). After exposure to ovalbumin, respiratory rate was least affected in positive control group. Respiratory rate was also less affected in test groups receiving MOLE at both dose levels (250 mg/kg and 500 mg/kg). 3.10. Effects on respiratory rate (Acetylcholine exposure/Ach) Ach exposure resulted in increased respiratory rate in diseased group (DC). Respiratory rate was less affected in groups receiving standard (OVA + DEX) and test drugs (OVA + MOLE). Respiratory rate was least affected in positive control group followed by test groups receiving MOLE at both dose levels (250 mg/kg and 500 mg/kg) (Fig. 10).
3.11. Effect of MOLE on tidal volume (Ovalbumin exposure)
Fig. 1. TLC plates visualised under UV/visible spectrophotometer at (A) 254 nm, (B) 366 nm, and (C) 530 nm respectively.
Tidal volume of animals was reduced in diseased group after exposure of animals to ovalbumin. Groups receiving standard (OVA + DEX) and test drugs (OVA + MOLE) showed increased tidal volume in comparison to the diseased group (DC) (Fig. 11). Maximum tidal volume was observed in case of group receiving standard drug followed by test groups receiving MOLE at both dose levels (250 mg/ kg and 500 mg/kg).
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Fig. 2. Chromatograms obtained after HPTLC analysis of TLC plates at (A) 254 nm (B) 366 nm and (C) 530 nm respectively.
3.12. Effect of MOLE on tidal volume (Acetylcholine exposure/Ach)
3.14. Histopathological analysis
Tidal volume was reduced significantly in diseased group (DC) after exposure to Ach. Reduction in tidal volume was less in groups receiving standard and test drugs. Tidal volume was maximum in positive control group receiving standard drug (OVA+DEX) followed by test groups receiving MOLE (OVA + MOLE) at both dose levels (250 mg/kg and 500 mg/kg) (Fig. 12).
Histopathology of lung tissues taken from animals belonging to different groups suggests a protective effect of MOLE. Lung tissue from diseased group (OVA exposed) showed characteristic thickening of basement membrane and airway smooth muscle. A high leucocyte count in the sub mucosal layer was evident from the microscopic images of the stained tissue. Thin basement membrane and airway smooth muscle with low leucocyte count was noticeable in case of positive control (OVA + DEX) and test groups (OVA + MOLE) (Fig. 13).
Table 1 Rf values at different wavelength 254 nm, 366 nm and 530 nm. Peak no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
4. Discussions and conclusions
Rf values 254 nm
366 nm
530 nm
0.10 0.16 0.62 0.65 0.68 0.74 0.98
0.13 0.17 0.41 0.49 0.52 0.64 0.81 0.88 0.96
0.05 0.07 0.13 0.18 0.24 0.27 0.31 0.41 0.50 0.61 0.64 0.72 0.78 0.81 0.87 0.92 0.98
In context of the pursuit for therapeutically active agents against hypersensitive reactions that can be identified and isolated from herbals and medicinal plants that are traditionally utilized for the treatment of hypersensitive and allergic reactions and to consolidate 0.8 y = 0.0023x + 0.0177 A 0.6 R² = 0.9999 b s n 0.4 o c r e 0.2 b 0 a 0 100 200
Series1 Linear (Series1) 300
400
Concentraon (μg/ ml) Fig. 3. Standard curve of Gallic Acid.
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Table 2 Concentration of total phenolics present in MOLE. Plant extract MOLE
Absorbance 0.212
0.261
0.263
Mean absorbance
Concentration (mg/ml)
Standard deviation
Mean Conc. (mg/ml)
0.245
8.4
1.2
9.8
10.5
10.6
Fig. 4. Standard curve of Catechin.
Table 3 Concentration of flavonoids present in MOLE. Plant extract MOLE
Absorbance 0.16
0.20
0.16
Mean absorbance
Concentration (mg/ml)
Standard deviation
Mean Conc. (mg/ml)
0.178
0.44
0.046
0.46
0.52
0.44
500
DAY 0 DAY 7
400
DAY 14
300
DAY 21
200 100
) g /k g
) (5 00 m
g /k g L E M
O
L E O M
O
V
A +D E
X
(2 50 m
(2 .5 m
N
D
g /k g
C
)
0
C
Bodyweight(gms)
Mean body weight
Groups Fig. 5. Histogram representing the effects of standard drug (OVA + DXM) and test drugs (OVA + MOLE) on body weights of guinea pigs. Data is expressed as mean § SEM. n = 6/group.
the suitability of medicinal plants in the treatment of these disorders, we carried out the safety and efficacy evaluation of leaves of a traditionally utilized plant Moringa oleifera for the treatment of Asthma in ovalbumin sensitized guinea pig. To study the phytochemical composition of the extract, we carried out phytochemical screening of the MOLE extract that showed the presence of flavonoids, glycosides, tannins, phenolics and saponins. A similar study conducted by
Vergara-Jimenez et al. (2017) also showed that the plant is rich in these phytoconstituents. A significant anti-inflammatory effect of plant was also evident from the earlier study (Muchirah et al., 2018). HPTLC analysis of the MOLE showed a number of bands when visualized under 254 nm, 366 nm and 530 nm. A similar study conducted by Makita et al. (2016) also showed the presence of several peaks and areas under HPTLC analysis suggesting a number of different
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WBC WBCCount103/µL
20 15 10 5
(2 D .5 C L m E g ( / M 25 kg O 0 ) L m E g (5 /k 0 g 0m ) g /k g ) O
M
O
V
A +
D E
X
N
C
0
Groups Fig. 7. Histogram representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on leucocyte count in BAL fluid. Data is represented as mean § SEM. n = 6/group.
Histamine level in lung tissue 15
(µg/goflungtissue)
Histamine concentration
Fig. 6. Histogram representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on total leucocyte count of guinea pigs. Data represented as mean § SEM. n = 6/group. * represents significant difference in leucocyte counts of treatment groups to that of disease group (p < 0.05).
Histamine level
10
*
*
5
(2 D .5 C LE m g M (25 /kg O 0 ) LE m g/ (5 00 kg) m g/ kg ) O
M
O
V
A +D E
X
N
C
0
Groups Fig. 8. Histogram representing the effects of standard drug (OVA + DEX) and test drug (OVA + MOLE) on histamine level (mg/g). All bars represent the mean § SEM. n = 6/group.*represents significant difference in histamine levels in treated groups to that of disease group (p < 0.05).
phytoconstituents present in the extract. Photometric estimation of plant extract showed the presence of an appreciable quantity of Phenolic is 9.8 g equivalent of Gallic acid and flavonoid 0.46 g equivalent of Catechin (Olszewska, 2007). The present work was carried out to examine the effect of MOLE in ovalbumin-induced airway inflammation in guinea pigs to reveal its potential against the symptoms of allergic asthma. An increased number of WBC count in whole blood as well as in BAL fluid of OVA control was evident in diseased animals after sensitization as compared to normal control. The reduction in WBC count was observed in standard and test drug (MOLE 250 mg/kg and 500 mg/kg). Appreciable results were observed in animals administered with both doses of extract. There were no fluctuations in body weights that were observed during the course of the study suggesting that the extract caused no severe toxicity to animals. Histamine stored mainly in mast cells and basophils, is a prominent contributor to allergic disease. Elevations in plasma or tissue histamine levels have been noted during anaphylaxis and experimental allergic responses of the skin, nose, and airways. Of the four cardinal signs of
asthma (bronchospasm, edema, inflammation, and mucus secretion), histamine is capable of mediating the first two through its H1 receptor and mucus secretion through its H2 receptor. In the nose, mucus secretion can be reflexively mediated by H1 and possibly also by H2 receptors (White, 1990).
Table 4 Tabular data representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on histamine levels. Values are expressed as mean § SEM (n = 6/group). represents the significant difference in histamine levels of treated group to that of treatment group (p<0.05). Treatment group NC DC OVA+DEX (2.5 mg/kg) MOLE (250 mg/kg) MOLE (500 mg/kg)
Histamine (mg/gm of tissue) 4.53 § 0.63 11 § 0.34 6.1 § 0.60 6.89 § 0.70* 7.2 § 0.70*
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Respiratory rate
Tidal volume
PRE
*
*
POST
150 100 50
PRE
4
Tidal volume ml/sec
POST 3
*
2
*
1
g/ kg LE ) (2 50 m M g/ O kg LE ) (5 00 m g/ kg ) O
O
M
VA +D EX
O
M
O
VA +D EX
(2 .5 m
(2 .5 m
N
C
g/ kg LE ) (2 50 m M g/ O kg LE ) (5 00 m g/ kg )
D
C
C N
C
0
0
D
Respiratory rate (f) breathe per minute
200
491
Groups
Groups Fig. 9. Histogram representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on respiratory rate of guinea pigs (Ovalbumin exposure). Data is expressed as mean § SEM (n = 6/group). *represents significant difference in respiratory rates of diseased and treatment groups.
Fig. 11. Histogram representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on Tidal volume (Ovalbumin exposure) of guinea pigs. Data is expressed as mean § SEM (n = 6/group). *represents significant difference in the values of tidal volume of treated animals to that of disease control group (*p < 0.05).
With regards to effects on inflammatory cells that increase in numbers during the host response to the antigen, histamine has been shown to be released. In the present study the histamine level was increased manifold in diseased group in comparison to normal control. The histamine content was significantly decreased in treatment groups receiving standard and test drug of MOLE (250 mg/kg and 500 mg/kg). Both dose of extract was found to be effective after the standard drug dexamethasone. Lung function test and Bronchoconstriction test in disease control animals showed decrease in their tidal volume in comparison to treatment group. A bronchodilatory effect of MOLE at dose 250 mg/kg and 500 mg/kg was observed. The potential mode of action employed by MOLE can be attributed to its bronchodilatory effect, ability of block the release of inflammatory mediators into the local lung tissues. An appreciable anti-inflammatory activity of MOLE was also inferable from the another study (Deori et al.,
2017). In respiratory rate both tests were applied and the disease control animals showed increase in their respiratory rate that was indicative of exertional breathing, a symptom of asthma. Further, the extract and standard compound showed bronchodilatory effect. Moreover, MOLE-treatment showed protection against bronchoconstriction and airway inflammation which was confirmed by histopathological observation. The present study on Moringa oleifera leaves extract (MOLE) shows that it possesses a potential anti-asthmatic activity and can be further explored to understand the molecular mechanism behind its anti-asthmatic activity. Further study on active phytoconstituents in the extract can be helpful in the anti- asthmatic research. In conclusion our data suggest that Moringa oleifera leaf produced beneficial effects against inflammation, bronchospasm, mast cell degranulation, immune reactions and anaphylactic reactions. This extract was found to inhibit the inflammatory mediator, histamine. Evidences from the present study this confirm the anti-asthmatic activity of Moringa oleifera leaves.
Respiratory rate (Ach) PRE POST *
Respiratory rate (f) breathe per minute
200
*
150 100 50
g/ kg LE ) (2 50 m M g/ O kg LE ) (5 00 m g/ kg )
C D
O
M
O
VA +D EX
(2 .5 m
N
C
0
Groups Fig. 10. Histogram representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on respiratory rates (Acetylcholine exposure/Ach) of guinea pigs. Data is expressed as mean § SEM (n = 6/group). * represents significant difference in respiratory rate of animals belonging to treatment groups to that of diseased group (*p < 0.05).
Fig. 12. Histogram representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on Tidal volume (Acetylcholine exposure/Ach) of guinea pigs. Data is expressed as mean § SEM (n = 6/group). *represents significant difference in tidal volume of treated animals to that of disease group (DC) (*p < 0.05).
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Fig. 13. Histopathology of lung tissue of animals treated with MOLE (Magnification 40X). Lung tissue of treated and untreated (OVA-sensitized) animals stained with haematoxylineosin. (A): Normal lung tissue, (B): typical damaged lung tissue from OVA-control (disease group), arrow shows thickened airway smooth muscle, thick basement membrane and high leucocyte count infiltrate in sub-mucosal layer, (C): Section from OVA + DEX (2.5 mg/kg) positive control and the arrow shows less leucocyte infiltration, (D): Section from OVA +MOLE (250 mg/kg) treatment group, (E): Section from OVA +MOLE (500 mg/kg) treatment group. In Figure D and E arrows show significant protection against leucocyte infiltration, thin airway smooth muscle and thin basement membrane.
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References
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Availability of data and materials The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Declaration of Competing Interest The authors declare no conflicts of interest. CRediT authorship contribution statement Sandhya Suresh: Conceptualization, Writing - original draft. Abu Sufiyan Chhipa: Conceptualization, Writing - original draft. Mansi Gupta: Methodology. Sunali Lalotra: Methodology. S.S. Sisodia: Project administration. Ruma Baksi: Methodology. Manish Nivsarkar: Project administration. Acknowledgements The authors would like to thank B.V. Patel PERD center, Ahmedabad for providing all the facilities to conduct the experiments. Funding None to declare.
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Phytochemical analysis and pharmacological evaluation of methanolic leaf extract of Moringa oleifera Lam. in ovalbumin induced allergic asthma Sandhya Suresha,b,1, Abu Sufiyan Chhipaa,b,1,*, Mansi Guptaa,b, Sunali Lalotraa,b, S.S. Sisodiab, Ruma Baksia,c, Manish Nivsarkara a
Department of Pharmacology and Toxicolcogy, B.V. Patel PERD Centre, Ahmedabad, Gujarat, India Faculty of Pharmacy, Bhupal Noble’s University, Udaipur, Rajasthan, India c Registered PhD scholar (External) at Faculty of Science, NIRMA University, Sarkhej-Gandhinagar Highway, Ahmedabad, Gujarat 382481, India b
A R T I C L E
I N F O
Article History: Received 16 July 2019 Revised 25 December 2019 Accepted 30 January 2020 Available online xxx Edited by AR Ndhlala Keywords: Asthma Moringa oleifera MOLE Bronchoconstriction Histamine
A B S T R A C T
Aim of the study: The present study was carried out to evaluate the anti-asthmatic effects of Moringa oleifera Lam. leaf extract (MOLE) on ovalbumin-induced asthma in guinea pigs. Materials and method: Extraction, preliminary phytochemical screening and HPTLC fingerprinting analysis of MOLE was carried out. Total phenolics and flavonoids in the extract were determined. Evaluation of antiasthmatic effect of the extract was done in guinea pigs sensitized with OVA albumin. Dexamathasone was taken as standard drug, while the test extract was administered at two dose levels before challenging the experimental animals with aerosolized 0.5% OVA albumin. Animals were randomized and divided into 5 groups namely normal control (NC), disease control (DC), positive control (PC), Test group 1 (T-1) and Test group 2 (T-2). Bronchoconstriction and lung function tests were performed. At the end of study, blood was collected from animals in each group to take total WBC count. Leucocyte count was also taken from Bronchoalveolar lavage fluid (BAL) collected from the lungs of animals belonging to each group. Histamine estimation in lung tissues was performed. Changes in lung tissue were observed by histopathological study. Results: Extractive value of MOLE was 24.9%. Phytochemical screening of extract suggested that the extract is rich in broad classes of phytoconstituents including flavonoids, phenolics, glycosides and tannins. Fingerprint analysis was conclusive of the presence of multiple compounds in the extract. Total phenolics in the extract were found to be 9.8 g equivalent of gallic acid while the flavonoidal content was 0.46 g equivalent of Catechin. Administration of standard and test extracts resulted in improved lung functions. Conclusion: Methanolic extract of Moringa oleifera leaf has beneficial effects against bronchoconstriction, airway inflammation and asthma. Further exploratory studies may lead to identification and isolation of potential anti-asthmatic candidate molecules from the plant. © 2020 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Asthma is a chronic inflammatory disease characterized by increased responsiveness of trachea and bronchi to multiple stimuli. This allergic disease manifests as airway obstructions that involves inflammation of the pulmonary airways and bronchial hyper responsiveness (Education et al., 2007; Shifren et al., 2012). Various inflammatory cells come into play during asthma and bronchoconstriction. These include eosinophils, lymphocytes, neutrophils monocytes, histamine, cytokines, leukotriene, prostaglandins and thromboxanes (Taur and Patil, 2011b; Pedersen et al., 1996; Andrade and de Sousa,
* Corresponding author at: 17, Chhipa Bakhal, Khanpura, Mandsaur, Madhya Pradesh 458001, India. E-mail address: asufi[email protected] (A.S. Chhipa). 1 Authors contributed equally. https://doi.org/10.1016/j.sajb.2020.01.046 0254-6299/© 2020 SAAB. Published by Elsevier B.V. All rights reserved.
2013; Kapoor et al., 2011; Wang et al., 2016; Machado-Carvalho et al., 2014; Arimura et al., 1992; Okechukwu and Ekeuku, 2012). The associated side effects of currently available medications for the treatment of asthma makes it imperative for the researchers to search for more effective drugs with least or no side effects. Herbal medicines are promising alternatives to these interventions owing to their acceptability among patients, efficacy and least side effects (Patel et al., 2020). Medicinal plants with significant anti-inflammatory, immunomodulatory, antihistaminic and smooth-muscle relaxant activity are proved to be beneficial in the management of asthma (Taur and Patil, 2011a). Natural products from plants sources have been utilized for the alleviation of human illness since the ancient times (Chhipa and Sisodia, 2019). Isolation and identification of flavonoids, glycosides, tannins, alkaloids, saponins and other phytochemicals from plant origin have been useful in controlling various inflammatory and allergic
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responses (Lee et al., 2011; Kimura et al., 1987; Khan, 2015; Zdanowicz, 2007; Sung et al., 2017). Flavonoids such as quercetin, rutin, apigenin and kaempferol are known contributors in the prevention of airway inflammatory responses and asthma (Li et al., 2010; Jung et al., 2007; Wang et al., 2018). Moringa oleifera Lam. is a drought resistant tree that belongs to family Moringaceae (Kou et al., 2018). It is widely utilized traditionally for its effectiveness against wide range of diseases and disorders including hyperglycaemia, lithiasis, hypertension, microbial, fungal viral infections, hepatotoxicity, inflammation and fever. Importantly, the plant parts are also utilized in the treatment of asthma (Rao et al., 1999; Denny Fitriana et al., 2016; Ingale and Gandhi, 2016; El-bakry et al., 2016; Khan et al., 2017; Mishra et al., 2011). The plant also has recognition in ayurvedic and unani systems of medicine (Mughal et al., 1999). Different parts of the plant are found to be rich in a plethora of phytoconstituents. These include alkaloids, flavonoids, tannins, quinine, saponins, glycosides and fixed oils (Paikra et al., 2017). However, these phytoconstituents are found to be nonuniformly present in different parts of the plant. For instance, flavonoidal glycosides (quercetin, kaempferol and isorhamnetin) are present mostly in leaves while the roots and seeds are devoid of flavonoidal contents (Saini et al., 2016; Aja et al., 2014; Vergara-Jimenez et al., 2017; Makita et al., 2016; Sulastri et al., 2018b; Lin et al., 2018). Moreover, previous studies on phytochemical screening of the seeds and leaves of the plant suggest that the leaves carry higher phytochemical composition in comparison to the seeds of Moringa Oleifera (Aja et al., 2014; Vergara-Jimenez et al., 2017). Preclinical studies have showed significant anti-inflammatory, anaphylactic and antiasthmatic effects of the seeds of Moringa oleifera (Mahajan et al., 2007; Agrawal and Mehta, 2008; Mahajan and Mehta, 2008b). However, the leaves of the plant are less studied in terms of their anti-inflammatory and anti-asthmatic effects. Moreover, based on the phytochemical composition of leaves which is much higher than the seeds of the plant (Aja et al., 2014; Vergara-Jimenez et al., 2017), higher efficiency of leaves against inflammation and asthmatic responses is undoubtedly perceivable. Based on these findings, we have attempted to evaluate the anti-asthmatic effects of methanolic extract of Moringa oleifera leaves in ova-albumin induced airway inflammation and asthma in guinea pigs. The extract was tested at two dose levels (250 mg/kg and 500 mg/kg). Dexamethasone is an effective anti-asthmatic agent that is used to treat asthma (Cross et al., 2011; Shefrin and Goldman, 2009). It is a corticosteroid drug that exerts its action by suppressing the late phase inflammation and chemotaxis in asthma (Townley and Suliaman, 1987). Based on the previous reports of the plant as an anti-inflammatory and antianaphylactic agent, we have used dexamethasone as the standard drug to compare the efficacy of the leaf extract with standard interventions in controlling airway inflammation and asthmatic responses. Moreover, to evaluate the effects of the extract in the alleviation of human allergic asthma, guinea pigs sensitized with ovalbumin allergen is a preferred animal model. In that view, the present study was conducted in ovalbumin sensitized guinea pigs to explore the potential of methanolic extract of leaves of Moringa oleifera in controlling airway inflammation and asthma. 2. Materials and methods 2.1. Reagents Methanol, Dragondroff’s reagent, chloroform, Sodium hydroxide, sodium sulphate, Ferric chloride, copper sulphate, toluene, ethyl acetate, Folin Coicalteu reagent, sodium carbonate, Gallic acid, Sodium nitroxide, Aluminium trichloride, perchloric acid, hydrochloric acid were obtained from the department of Pharmacology and Toxicology, B.V. Patel PERD center. Dexamethasone, acetylcholine and histamine were purchased from MP biomedicals. Aluminum hydroxide, catechin, Agar, Formaldehyde and Sodium chloride were purchased from
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fisher scientific. n-Butanol and n-Heptane were purchased from Merck specialities Pvt. Ltd. Isoflurane and Ovalbumin were purchased from Raman and well Pvt. Ltd. and HIMEDIA respectively. All chemicals and reagents were of analytical grade. 2.2. Procurement and identification of the plant material Leaves of Moringa oleifera Lam. were collected from the medicinal plant garden of B.V. Patel PERD center, Ahmedabad in the month of October 2018. Plant material was identified and authenticated by taxonomist in Department of Pharmacognosy and Phytochemistry, B.V. PERD center. A voucher specimen was submitted in the department for future reference. Voucher specimen number: BVPPERD/PP/1118/08. 2.3. Extraction of plant material Moringa oleifera leaves were shade dried for 1 week at 37°C. Dried leaves were powdered and the weight of coarse material was taken. For extraction, 350 g material was taken in 1litre of methanol in the conical flasks in two sets such that each flask receives 500 ml of methanol. Flasks were kept overnight. After 3 days the solutions in the flasks were filtered and kept on a rotary evaporator at 50°C to evaporate the solvent. The process was repeated 3 times with the remaining marc. The extract obtained after evaporation was combined and percent yield of Moringa oleifera leaf extract (MOLE) was calculated (Vongsak et al., 2013; Mahdi et al., 2016). 2.4. Preliminary phytochemical screening of extract Preliminary phytochemical analysis of extract was performed for the identification of broad classes of phytoconstituents including alkaloid, steroid, flavonoid, phenolics, glycosides, tannins and saponins according to their standard method (Ezeonu and Ejikeme, 2016; Dahiru et al., 2006). 2.5. Phytochemical fingerprinting analysis High performance Thin Layer Chromatography (HPTLC) was done for fingerprinting analysis of the extract using Hexane: Ethyl acetate: Acetic acid as the solvent system taken in the ratio 5:4:1. Solvent system was prepared (10 ml) and kept for 20 min in the TLC chamber for saturation to take place. For HPTLC analysis, the test solution (15 ng) was applied in the form of band (8 mm) by using 100 mL of CAMAG syringe on 10 £ 10 cm aluminium packed TLC plate with the help of a 100mL Hamilton syringe and Linomat V applicator attached with HPTLC system programmed through Win CATS software. TLC plates were developed by the ascending technique using 10 ml of mobile phase [Hexane: Ethyl acetate: Acetic acid] taken in the ratio 5:4:1 in a CAMAG twin-through glass chamber and saturated with mobile phase covered with a stainless-steel lid. TLC plate was dried. The image of the plate was taken in daylight and then in photo-documentation chamber. Images were captured at 254 nm, 366 nm and 530 nm (after derivatisation with Anisaldehyde Sulphuric acid reagent). Densitometric scanning was performed with scanner equipped with Win CATS package at λmax = 254, 366 and 530 nm. Peak tables, Rf values, and HPTLC chromatograms were recorded (Swathi, 2016; Elangovan et al., 2015). 2.6. Estimation of total phenolic contents by spectrophotometry Powdered material (Moringa oleifera leaf) of the plant was weighed and extracted with 10 ml of 50% aqueous methanol and the solution was filtered and collected. The final volume was made up with 50 ml methanol. From this, 0.1 ml solution was taken and added in 10 ml of double distilled water and 1.5 ml of Folin-Ciocalteu reagent in a 25 ml volumetric flask followed by incubation for 5 min.
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4 ml of 20% sodium carbonate was added and the final volume was made to 25 ml with double distilled water. Solution was mixed thoroughly and incubated at room temperature for 30 min. The absorption was recorded at 765 nm. Gallic acid (100 mg/ml) was used as the standard phenolic (Sulastri et al., 2018a; Tabasum et al., 2016). 2.7. Estimation of total flavonoids contents by photometry For the estimation of total flavonoids in the extract, 125mL of extract solution in methanol was taken and 75 mL of 5% NaNO2 solution was added in it. The mixture was allowed to stand for 6 min. followed by addition of 150mL of aluminium trichloride (10%) and incubation for 5 min. Thereafter, 750mL of NaOH (1 M) was added to the mixture and final volume of the solution was adjusted to 2.5 ml with distilled water. The mixture was allowed to turn pink and final the absorbance was measured at 510 nm. Catechin was taken as standard flavonoid and total flavonoids content was expressed as gram et al., 2018). equivalent of catechin (Okumu et al., 2016; Nobosse 2.8. Animal husbandry and feeds Dunkin-Hartley guinea pigs (350 450 g) of either sex were housed in a room maintained at 25 °C with a relative humidity of 65% in 14-hr light-dark cycle. Nutritional solutions and green grass were given ad libitum to the animals and filtered tap water was kept for drinking. All experiments were performed under ethical guidelines. The protocol was reviewed and approved by the Institutional animal ethics committee (IAEC), Protocol no: PERD/IAEC/2018/015. Through the entire experimental period, growth, health, and food intake capacity of animals were monitored and maintained. 2.9. Sensitization and treatment of animals Animals were randomised in five different groups (n = 6/group). Group I: normal control (NC) receiving agar solution, Group II disease control (DC) (Ovalbumin sensitized), Group III positive control (PC) receiving dexamethasone (Ovalbumin sensitized then received 2.5 mg/kg dexamethasone according to their body weights), Group IV test control (T-1) receiving Moringa oleifera leaf extract (Ovalbumin sensitized then received 250 mg/kg MOLE according to body weight), group V test control (T-2) receiving Moringa oleifera leaf extract (Ovalbumin sensitized then received 500 mg/kg MOLE according to body weight. All selected animals (except group I) were sensitized with 100 mg of ovalbumin absorbed into aluminium hydroxide saline solution by using a subcutaneous injection on day 0 as the first sensitization. Animals were sensitized for the second time on day 14. Dosing of the test drug and positive control are started from day 14 to 21, one hour prior to sensitization (Mahajan and Mehta, 2008a). 2.10. Ovalbumin exposure On day 18 to day 21, animals in each group (except group I) were challenged with aerosolized 0.5% OVA for 2 min., 2.5 h after receiving their drug or vehicle treatment. For the challenge, conscious animals were placed into a plastic circular chamber (diameter = 70 cm, height = 40 cm) connected to a nebulizer (CX4-Omron Healthcare Company Ltd. Kyoto, Japan). Animals in the group I were exposed to aerosolized saline (Mahajan and Mehta, 2008a; Meurs et al., 2006). 2.11. Haematology On day 17 and 22, blood from animals in each group was collected and total leucocyte count was measured in an automated haematology analyser (VetScan HM-5; Abaxis Inc., Union City, CA, USA) (31).
2.12. Measurement of lung function test On day 21, animals from each group were subjected to lung function parameter measurements. Tidal volume and respiratory rate in guinea pigs were measured before and after the aerosolized ovalbumin exposure with the help of nebulizer (OMRON, NE C-25 model) and recordings were taken in the biopac system (model MP-35, Biopac System, Inc., Santa Barbara, CA). Animals in group I (normal control) animals were exposed to aerosolized saline (Mahajan and Mehta, 2008a). 2.13. Measurement of bronchoconstriction test After the measurement of the lung function parameters, all animals were subjected to a bronchoconstriction test. OVA-sensitized hosts were exposed (in a conscious state) to 0.25% acetylcholine (ACH) solution for 30 s using a nebulizer (OMRON, NE C-25 model.) connected to the animal holder. Guinea pigs in the normal control group were exposed to normal saline in place of Ach. In each case, before and after the exposure of Ach, the tidal volume and respiration rate of each animal was noted (Mahajan and Mehta, 2008a). 2.14. Collection of bronchoalveolar lavage (BAL) fluid For the collection of BAL fluid, guinea pigs were anesthetized by isoflurane in the anaesthesia chamber. A polypropylene cannula was inserted into the trachea and 0.9% (w/v) normal saline solution (10 ml) was introduced into the lungs using a 10-ml syringe. The injected saline was recovered 5 min later. The recovered lavage fluid (5 ml) was centrifuged at 5000 x g for 10 min at 4 °C; the resultant supernatant was discarded and the cells in the pellet were washed in 0.5 ml saline and total cell counts were then performed in Automated haematology analyser (VetScan HM-5; Abaxis Inc., Union City, CA, USA) (Mahajan and Mehta, 2008a). 2.15. Histamine assay in lung tissue Lung tissues from animals belonging to different groups were collected and used for the estimation of histamine. Around 200 g of lung tissue was mixed with 2.5 ml normal saline and 2.5 ml of 0.8 N perchloric acid followed by centrifugation (4000 £ g, 7 min at 4 °C). The resulting supernatant (2 ml) was taken in a test tube containing 0.25 ml of 5 N NaOH, 0.75 g NaCl, and 5 ml n-butanol. The mixture was then was vortexed for 5 min to ensure the histamine localization into the butanol followed by centrifugation. The aqueous phase after centrifugation was discarded while the organic phase was washed with 2.5 ml salt-saturated 0.1 N NaOH solution to ensure the removal of any residual histidine. Thereafter, the mixture was centrifuged again and butanol was taken to a test tube containing 2 ml of 0.1 N HCl and 5 ml n-heptane. The mixture was again centrifuged and the presence of histamine determined fluorometrically (Shore et al., 1959; Jannatin et al., 2017). 2.16. Histopathological analysis Histopathology analysis of lung tissues obtained from each guinea pig was carried out to study the effect of extract on the immunemediated chronic inflammation brought about by sensitization with OVA. Dissected lungs and trachea were washed with normal saline and then placed in a 10% formaldehyde solution for 1week. After the tissues were fixed, specimens of lung tissues were embedded in paraffin wax and 5pm sections were cut and stained with haematoxylin and eosin dye for morphology. Images of selected sections were captured at 40X magnifications using a zoom digital camera optical microscopy (IX 51; Olympus, Tokyo, Japan) equipped with a digital camera (TL4) (Mahajan and Mehta, 2008a).
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2.17. Statistical analysis
3.5. Effect of MOLE on body weight
Results were reported as mean § SEM. Statistical analysis was performed using one-way analysis of variance ANOVA and t-test using graph pad prism 8 software. The data was considered statistically significant at p < 0.05.
All animals in diseased and treatment groups (Positive and test control) did not show any significant difference in body weight during experimental period in comparison to those present in normal control group. The result is suggestive that MOLE did not interfere with normal growth of animals (Fig. 5).
3. Results 3.1. Extraction and preliminary phytochemical study The percent yield of methanolic extract of Moringa oleifera leaf (MOLE) was found to be 24.9%. The preliminary phytochemical screening of MOLE showed the presence of flavonoids, glycosides, tannins, and phenolics.
3.6. Effect of MOLE on leucocyte count in the blood WBC counts were significantly different among different groups. The administration of standard drug OVA+DEX and extract at both dose levels (MOLE 250 mg/kg and MOLE 500 mg/kg) lowered the leucocyte count significantly as compared to disease group (*p < 0.05) on days 17 and 22. Reduction in WBC count was maximum in positive control group receiving standard drug (OVA+DXM) followed by test groups receiving MOLE (250 mg/kg and 500 mg/kg) (Fig. 6).
3.2. HPTLC fingerprinting analysis Peaks corresponding to various phytoconstituents were obtained. TLC plate under CAMAG HPTLC system resulted in three different chromatograms at 254 nm, 366 nm and 530 nm at 7, 10 and 17 peaks respectively (Figs. 1 and 2). Ranges of Rf values were in between 0.10 0.98, 0.13 0.96 and 0.05 0.98 under these wavelength (Table 1).
3.7. Effect of MOLE on leucocyte count in BAL fluid All groups receiving interventions (Positive control and test groups) showed significant reduction in WBC in comparison to disease group (p<0.05) (Fig. 7). However, maximum reduction in WBC counts was observed in positive control (OVA+DXM) followed by test groups receiving higher dose (500 mg/kg) and lower dose (250 mg/ kg) of MOLE.
3.3. Spectrophotometric estimation of total phenolic contents 3.8. Effect of MOLE on histamine level Total phenolics in the extract were calculated from the standard curve of gallic acid (Fig. 3). Concentration of total phenolic was found to be 9.8 mg/ ml equivalent of Gallic acid (Table 2).
3.4. Spectrophotometric estimation of total flavonoid Flavonoid contents in the extract were obtained from the standard curve of Catechin (Fig. 4). Total flavonoids in the extract were found to be 0.46 mg/ml (Table 3).
Similar results were obtained in case of histamine levels in different groups. Maximum reduction in histamine levels was noticed in positive control group (OVA+DEX) (Fig. 8). Test groups receiving the MOLE at both dose levels (250 mg/kg and 500 mg/kg) also showed significant reduction in histamine levels in comparison to diseased group (*p < 0.05) (Table 4). 3.9. Effects on respiratory rate (Ovalbumin exposure) Respiratory rate was less in groups receiving standard (OVA + DEX) and test drugs (OVA + MOLE) (Fig. 9). After exposure to ovalbumin, respiratory rate was least affected in positive control group. Respiratory rate was also less affected in test groups receiving MOLE at both dose levels (250 mg/kg and 500 mg/kg). 3.10. Effects on respiratory rate (Acetylcholine exposure/Ach) Ach exposure resulted in increased respiratory rate in diseased group (DC). Respiratory rate was less affected in groups receiving standard (OVA + DEX) and test drugs (OVA + MOLE). Respiratory rate was least affected in positive control group followed by test groups receiving MOLE at both dose levels (250 mg/kg and 500 mg/kg) (Fig. 10).
3.11. Effect of MOLE on tidal volume (Ovalbumin exposure)
Fig. 1. TLC plates visualised under UV/visible spectrophotometer at (A) 254 nm, (B) 366 nm, and (C) 530 nm respectively.
Tidal volume of animals was reduced in diseased group after exposure of animals to ovalbumin. Groups receiving standard (OVA + DEX) and test drugs (OVA + MOLE) showed increased tidal volume in comparison to the diseased group (DC) (Fig. 11). Maximum tidal volume was observed in case of group receiving standard drug followed by test groups receiving MOLE at both dose levels (250 mg/ kg and 500 mg/kg).
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Fig. 2. Chromatograms obtained after HPTLC analysis of TLC plates at (A) 254 nm (B) 366 nm and (C) 530 nm respectively.
3.12. Effect of MOLE on tidal volume (Acetylcholine exposure/Ach)
3.14. Histopathological analysis
Tidal volume was reduced significantly in diseased group (DC) after exposure to Ach. Reduction in tidal volume was less in groups receiving standard and test drugs. Tidal volume was maximum in positive control group receiving standard drug (OVA+DEX) followed by test groups receiving MOLE (OVA + MOLE) at both dose levels (250 mg/kg and 500 mg/kg) (Fig. 12).
Histopathology of lung tissues taken from animals belonging to different groups suggests a protective effect of MOLE. Lung tissue from diseased group (OVA exposed) showed characteristic thickening of basement membrane and airway smooth muscle. A high leucocyte count in the sub mucosal layer was evident from the microscopic images of the stained tissue. Thin basement membrane and airway smooth muscle with low leucocyte count was noticeable in case of positive control (OVA + DEX) and test groups (OVA + MOLE) (Fig. 13).
Table 1 Rf values at different wavelength 254 nm, 366 nm and 530 nm. Peak no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
4. Discussions and conclusions
Rf values 254 nm
366 nm
530 nm
0.10 0.16 0.62 0.65 0.68 0.74 0.98
0.13 0.17 0.41 0.49 0.52 0.64 0.81 0.88 0.96
0.05 0.07 0.13 0.18 0.24 0.27 0.31 0.41 0.50 0.61 0.64 0.72 0.78 0.81 0.87 0.92 0.98
In context of the pursuit for therapeutically active agents against hypersensitive reactions that can be identified and isolated from herbals and medicinal plants that are traditionally utilized for the treatment of hypersensitive and allergic reactions and to consolidate 0.8 y = 0.0023x + 0.0177 A 0.6 R² = 0.9999 b s n 0.4 o c r e 0.2 b 0 a 0 100 200
Series1 Linear (Series1) 300
400
Concentraon (μg/ ml) Fig. 3. Standard curve of Gallic Acid.
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489
Table 2 Concentration of total phenolics present in MOLE. Plant extract MOLE
Absorbance 0.212
0.261
0.263
Mean absorbance
Concentration (mg/ml)
Standard deviation
Mean Conc. (mg/ml)
0.245
8.4
1.2
9.8
10.5
10.6
Fig. 4. Standard curve of Catechin.
Table 3 Concentration of flavonoids present in MOLE. Plant extract MOLE
Absorbance 0.16
0.20
0.16
Mean absorbance
Concentration (mg/ml)
Standard deviation
Mean Conc. (mg/ml)
0.178
0.44
0.046
0.46
0.52
0.44
500
DAY 0 DAY 7
400
DAY 14
300
DAY 21
200 100
) g /k g
) (5 00 m
g /k g L E M
O
L E O M
O
V
A +D E
X
(2 50 m
(2 .5 m
N
D
g /k g
C
)
0
C
Bodyweight(gms)
Mean body weight
Groups Fig. 5. Histogram representing the effects of standard drug (OVA + DXM) and test drugs (OVA + MOLE) on body weights of guinea pigs. Data is expressed as mean § SEM. n = 6/group.
the suitability of medicinal plants in the treatment of these disorders, we carried out the safety and efficacy evaluation of leaves of a traditionally utilized plant Moringa oleifera for the treatment of Asthma in ovalbumin sensitized guinea pig. To study the phytochemical composition of the extract, we carried out phytochemical screening of the MOLE extract that showed the presence of flavonoids, glycosides, tannins, phenolics and saponins. A similar study conducted by
Vergara-Jimenez et al. (2017) also showed that the plant is rich in these phytoconstituents. A significant anti-inflammatory effect of plant was also evident from the earlier study (Muchirah et al., 2018). HPTLC analysis of the MOLE showed a number of bands when visualized under 254 nm, 366 nm and 530 nm. A similar study conducted by Makita et al. (2016) also showed the presence of several peaks and areas under HPTLC analysis suggesting a number of different
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WBC WBCCount103/µL
20 15 10 5
(2 D .5 C L m E g ( / M 25 kg O 0 ) L m E g (5 /k 0 g 0m ) g /k g ) O
M
O
V
A +
D E
X
N
C
0
Groups Fig. 7. Histogram representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on leucocyte count in BAL fluid. Data is represented as mean § SEM. n = 6/group.
Histamine level in lung tissue 15
(µg/goflungtissue)
Histamine concentration
Fig. 6. Histogram representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on total leucocyte count of guinea pigs. Data represented as mean § SEM. n = 6/group. * represents significant difference in leucocyte counts of treatment groups to that of disease group (p < 0.05).
Histamine level
10
*
*
5
(2 D .5 C LE m g M (25 /kg O 0 ) LE m g/ (5 00 kg) m g/ kg ) O
M
O
V
A +D E
X
N
C
0
Groups Fig. 8. Histogram representing the effects of standard drug (OVA + DEX) and test drug (OVA + MOLE) on histamine level (mg/g). All bars represent the mean § SEM. n = 6/group.*represents significant difference in histamine levels in treated groups to that of disease group (p < 0.05).
phytoconstituents present in the extract. Photometric estimation of plant extract showed the presence of an appreciable quantity of Phenolic is 9.8 g equivalent of Gallic acid and flavonoid 0.46 g equivalent of Catechin (Olszewska, 2007). The present work was carried out to examine the effect of MOLE in ovalbumin-induced airway inflammation in guinea pigs to reveal its potential against the symptoms of allergic asthma. An increased number of WBC count in whole blood as well as in BAL fluid of OVA control was evident in diseased animals after sensitization as compared to normal control. The reduction in WBC count was observed in standard and test drug (MOLE 250 mg/kg and 500 mg/kg). Appreciable results were observed in animals administered with both doses of extract. There were no fluctuations in body weights that were observed during the course of the study suggesting that the extract caused no severe toxicity to animals. Histamine stored mainly in mast cells and basophils, is a prominent contributor to allergic disease. Elevations in plasma or tissue histamine levels have been noted during anaphylaxis and experimental allergic responses of the skin, nose, and airways. Of the four cardinal signs of
asthma (bronchospasm, edema, inflammation, and mucus secretion), histamine is capable of mediating the first two through its H1 receptor and mucus secretion through its H2 receptor. In the nose, mucus secretion can be reflexively mediated by H1 and possibly also by H2 receptors (White, 1990).
Table 4 Tabular data representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on histamine levels. Values are expressed as mean § SEM (n = 6/group). represents the significant difference in histamine levels of treated group to that of treatment group (p<0.05). Treatment group NC DC OVA+DEX (2.5 mg/kg) MOLE (250 mg/kg) MOLE (500 mg/kg)
Histamine (mg/gm of tissue) 4.53 § 0.63 11 § 0.34 6.1 § 0.60 6.89 § 0.70* 7.2 § 0.70*
S. Suresh et al. / South African Journal of Botany 130 (2020) 484 493
Respiratory rate
Tidal volume
PRE
*
*
POST
150 100 50
PRE
4
Tidal volume ml/sec
POST 3
*
2
*
1
g/ kg LE ) (2 50 m M g/ O kg LE ) (5 00 m g/ kg ) O
O
M
VA +D EX
O
M
O
VA +D EX
(2 .5 m
(2 .5 m
N
C
g/ kg LE ) (2 50 m M g/ O kg LE ) (5 00 m g/ kg )
D
C
C N
C
0
0
D
Respiratory rate (f) breathe per minute
200
491
Groups
Groups Fig. 9. Histogram representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on respiratory rate of guinea pigs (Ovalbumin exposure). Data is expressed as mean § SEM (n = 6/group). *represents significant difference in respiratory rates of diseased and treatment groups.
Fig. 11. Histogram representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on Tidal volume (Ovalbumin exposure) of guinea pigs. Data is expressed as mean § SEM (n = 6/group). *represents significant difference in the values of tidal volume of treated animals to that of disease control group (*p < 0.05).
With regards to effects on inflammatory cells that increase in numbers during the host response to the antigen, histamine has been shown to be released. In the present study the histamine level was increased manifold in diseased group in comparison to normal control. The histamine content was significantly decreased in treatment groups receiving standard and test drug of MOLE (250 mg/kg and 500 mg/kg). Both dose of extract was found to be effective after the standard drug dexamethasone. Lung function test and Bronchoconstriction test in disease control animals showed decrease in their tidal volume in comparison to treatment group. A bronchodilatory effect of MOLE at dose 250 mg/kg and 500 mg/kg was observed. The potential mode of action employed by MOLE can be attributed to its bronchodilatory effect, ability of block the release of inflammatory mediators into the local lung tissues. An appreciable anti-inflammatory activity of MOLE was also inferable from the another study (Deori et al.,
2017). In respiratory rate both tests were applied and the disease control animals showed increase in their respiratory rate that was indicative of exertional breathing, a symptom of asthma. Further, the extract and standard compound showed bronchodilatory effect. Moreover, MOLE-treatment showed protection against bronchoconstriction and airway inflammation which was confirmed by histopathological observation. The present study on Moringa oleifera leaves extract (MOLE) shows that it possesses a potential anti-asthmatic activity and can be further explored to understand the molecular mechanism behind its anti-asthmatic activity. Further study on active phytoconstituents in the extract can be helpful in the anti- asthmatic research. In conclusion our data suggest that Moringa oleifera leaf produced beneficial effects against inflammation, bronchospasm, mast cell degranulation, immune reactions and anaphylactic reactions. This extract was found to inhibit the inflammatory mediator, histamine. Evidences from the present study this confirm the anti-asthmatic activity of Moringa oleifera leaves.
Respiratory rate (Ach) PRE POST *
Respiratory rate (f) breathe per minute
200
*
150 100 50
g/ kg LE ) (2 50 m M g/ O kg LE ) (5 00 m g/ kg )
C D
O
M
O
VA +D EX
(2 .5 m
N
C
0
Groups Fig. 10. Histogram representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on respiratory rates (Acetylcholine exposure/Ach) of guinea pigs. Data is expressed as mean § SEM (n = 6/group). * represents significant difference in respiratory rate of animals belonging to treatment groups to that of diseased group (*p < 0.05).
Fig. 12. Histogram representing the effects of standard drug (OVA + DEX) and test drugs (OVA + MOLE) on Tidal volume (Acetylcholine exposure/Ach) of guinea pigs. Data is expressed as mean § SEM (n = 6/group). *represents significant difference in tidal volume of treated animals to that of disease group (DC) (*p < 0.05).
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Fig. 13. Histopathology of lung tissue of animals treated with MOLE (Magnification 40X). Lung tissue of treated and untreated (OVA-sensitized) animals stained with haematoxylineosin. (A): Normal lung tissue, (B): typical damaged lung tissue from OVA-control (disease group), arrow shows thickened airway smooth muscle, thick basement membrane and high leucocyte count infiltrate in sub-mucosal layer, (C): Section from OVA + DEX (2.5 mg/kg) positive control and the arrow shows less leucocyte infiltration, (D): Section from OVA +MOLE (250 mg/kg) treatment group, (E): Section from OVA +MOLE (500 mg/kg) treatment group. In Figure D and E arrows show significant protection against leucocyte infiltration, thin airway smooth muscle and thin basement membrane.
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References
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Availability of data and materials The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Declaration of Competing Interest The authors declare no conflicts of interest. CRediT authorship contribution statement Sandhya Suresh: Conceptualization, Writing - original draft. Abu Sufiyan Chhipa: Conceptualization, Writing - original draft. Mansi Gupta: Methodology. Sunali Lalotra: Methodology. S.S. Sisodia: Project administration. Ruma Baksi: Methodology. Manish Nivsarkar: Project administration. Acknowledgements The authors would like to thank B.V. Patel PERD center, Ahmedabad for providing all the facilities to conduct the experiments. Funding None to declare.
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