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Evaluation of potent phytomedicine for treatment of psoriasis using UV radiation induced psoriasis in rats
Biomedicine & Pharmacotherapy 84 (2016) 1156–1162
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Original article
Evaluation of potent phytomedicine for treatment of psoriasis using UV radiation induced psoriasis in rats Hemant K. Nagara,* , Amit K. Srivastavab , Rajnish Srivastavac, Mahendra S. Ranawata a
Bhupal Nobles' College of Pharmacy, Udaipur 313002, India Indian Institute of Integrative Medicine, (Council of Scientific & Industrial Research), Jammu- 180001, India c Moradabad Educational Trust, Group of Institutions, Faculty of Pharmacy, Moradabad 244001, India b
A R T I C L E I N F O
Article history: Received 10 August 2016 Received in revised form 8 October 2016 Accepted 9 October 2016 Keywords: UV radiation Psoriasis Woodfordia fructicosa Gardenia gummifera Severity index Epidermal thickness Hydroxyproline content
A B S T R A C T
The aim of present study was to determine the effect of newly formulated gels and suspensions of extractive Phytoconstituents of Woodfordia fructicosa flowers and Gardenia gummifera leaves by using UV Radiation induced psoriasis in rats. Both plants are traditionally claimed to be useful in treatment of number of skin diseases. However, there are no established scientific reports for their potential in psoriasis. Formulated Gels and Suspensions of ethanolic extract of both plants were tested for acute dermal and oral toxicity study respectively. The results of acute dermal toxicity at concentration 1% w/w and oral toxicity at dose 1000 mg/kg showed that the gels and suspensions were safe. Psoriasis was induced in Wistar rats by espousing 10% area of total body by UV radiations. Anti-psoriatic activity was performed by applying 0.1% gel and orally at a dose 100 mg/kg body weight in rats. Severity Index, histological study and biochemical estimation were analyzed. The results of our studies showed that the test formulations (Gels and Suspensions) of both plant extracts exhibited potential effect in anti-psoriatic activity. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Many herbal remedies individually or in combination have been recommended in various medical expositions for the cure of different diseases. Plants are among the most important and common sources of potentially valuable new drugs. Therefore, there is a need to investigate the biological properties of medicinal plants in order to develop new drugs. Much work has been carried out on herbal treatment of various diseases, but still there is need to explore more. Herbal remedies are promising in the management of dermatological conditions including psoriasis [1]. Psoriasis is a common skin disease affecting 2% of world population characterized by epidermal keratinocyte hyper proliferation, abnormal keratinocyte differentiation and immune cell infiltration [2–4]. Psoriasis is a recurrent and debilitating disease. The existing topical treatments such as emollients, coal tar and dithranol had lower efficacy and cosmetically unacceptable, while systemic therapies such as methotrexate, cyclosporine and acitretin had significant side effects [5]. Overexposure to ultraviolet (UV) radiation can cause a number of skin disorders such as erythema,
* Corresponding author. E-mail address: [email protected] (H.K. Nagar). http://dx.doi.org/10.1016/j.biopha.2016.10.027 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
edema, hyperpigmentation, immunosuppression, photoaging and skin cancer. Since the level of UV radiation is increasing as a result of depletion of the stratospheric ozone and climate change, the protection of human skin against the harmful effects of UV radiation is an urgent need [6]. Woodfordia fructicosa (Kurz.) family Lythraceae is a straggling leafy shrub, frequently used English names are Fire Flame Bush while locally known as Dhai is about 3.5 m in high and distributed abundantly throughout the North India, as well as in the majority of the East Asian countries. According to the Traditional system of medicine, the flowers of Woodfordia fructicosa are pungent, acrid, cooling, toxic, alexiteric, use as astringenttonic in disorders of mucous membranes, uterine sedative and anthelmintic and is useful in thirst, dysentery, leprosy, erysipelas, blood disease, leucorrhoea, menorrhagia and toothache [7,8]. In addition, Woodfordia fructicosa flowers are extensively used by tribal people for its wound, ulcer healing, analgesic and anti-rheumatic properties [9– 11]. Pharmacological claims of Woodfordia fructicosa dried flowers can be ascribed for its important bioactive phytoconstituents such as flavonoids, sterols, anthraquinones, saponins and tannins [12– 14]. Preclinical data from various studies indicates, that dried Woodfordia fructicosa flower extract possess anti-pyretic, Antiinflammatory [15], Anti-tumor [16,17], Anti-viral [18], Immunomodulatory [19], Anti-fertility [20], Antibacterial [21],
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Hepatoprotective [22], Anti-hyperlipedemic [23], Antidiabetic [24], Bronchoprotective [25], Wound healing [26] and Antiasthmatic activity [27]. Gardenia gummifera (family- Rubiaceae) is geographically distributed in India, Burma, Bangladesh, Konkan region, North Kanara, and Malabar Coast. G. gummifera is claimed to have a number of medicinal properties which include anthelmintic, antispasmodic, carminative, diaphoretic, expectorant, potentiation of pentobarbitone induced sleep, antiepileptic, peripheral and central analgesic, cardiotonic, antioxidant and anti-hyperlipidemic. It is also claimed to be useful in dyspepsia, flatulence for cleaning foul ulcers and wounds and to keep off flies from wounds in veterinary practice [28,29]. Dikamali is the gum resin obtained from the leaf buds of Gardenia gummifera [30]. A number of flavonoids such as gardenin A, B, C, D and E were isolated from the plant [31,32]. The close resemblance of inflammatory process produced by ultraviolet radiation to the one exhibited in psoriasis provides us with a good model to investigate drugs that have a potential to reduce the inflammatory reaction associated with psoriasis. Hence, Anti-psoriatic activity of phytomedicine extracted from Woodfordia fructicosa and Gardenia gummifera is evaluated using UV Radiation induced psoriasis in experimental rats. 2. Materials and methods 2.1. Chemicals Tretinoin Microsphere gel 0.04% w/w (Supatret 0.04) manufectured by sun pharmaceuticals Pvt. Ltd was purchased from medical store. Chloramine-T and Ehrlich reagent were purchased from Himedia laboratories Pvt. Ltd for hydroxyproline estimation. All other chemicals used for this study were of analytical grade. 2.2. Plant materials The flowers of Woodfordia fructicosa and leaves of Gardenia gummifera Linn were collected in the month of February 2014 from the Swarn Jayanti Park, near Sarvadharm colony, Bhopal (M.P.). Both the plant were authenticated by Dr. Zia Ul Hasan (Professor, Department of Botany), Safia College Bhopal. The voucher specimen for W. Fructicosa (460/Bot/Saif/14) and G. Gummifera (503/Bot/Saif/14) has been deposited in the herbarium section of Department of Botany, Safia College, Bhopal for future and further reference.
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Further required quantity of extract (100 mg for 0.1%) was mixed to the above mixture and volume made up to 100 ml by adding remaining distilled water. Finally full mixed ingredients were mixed properly to the Carbopol gel with continuous stirring and triethanolamine was added drop wise to the formulation for adjustment of required skin pH and to obtain the gel at required consistency. 2.4.2. Suspension Suspensions of extracts were prepared using 0.5% carboxyl methyl cellulose (CMC) as suspending agent. Calculated amount of extract was premixed in distilled water followed by addition of required amount of CMC, Sorbitol Solution (0.5%) and Methyl Paraben; the mixture was agitated with the help of glass rod and was finally sonicated with help of ultrasonicator. Suspensions of both extract were prepared to get the test doses (50 mg/kg per ml). 2.5. Animals care and acclimatization The animal experimental protocol was approved by the Institutional Animals Ethical Committee (IAEC), Sapience Bioanalytical Research Lab. (Reg. no. 1413/PO/E/S/11/CPCSEA), Bhopal, India. Protocol Approval Number is SBRL/IAEC/June 2014/14. Male & female Wistar albino rats (120–140 g) were provided by Sapience Bioanalytical Research Lab. The animals were housed in standard conditions of temperature (25 2 C) and 12:12 h light-dark cycle. The rats were fed with commercial diet and water ad Libitum. The animals were acclimatized to the laboratory conditions for a minimum period of seven days prior to commencement of treatment. 2.6. Acute dermal toxicity [33] The acute dermal toxicity test of extracts was determined according to the OECD guidelines no. 402. Adult Wistar rats of either sex were used. Nine animals were divided in three groups, each group comprises three animals. Approximately 24 h before the test, 10% hairs of the body were removed from the dorsal area of the test animals by using hair removal cream (Veet). Group I animals were considered as control, Group II and Group III animals received topically 1000 mg/kg body weight (limit test) of ethanolic extract of Woodfordia fructicosa flowers and Gardenia gummifera leaves respectively. All animals were monitored for 14 days for changes in fur, eyes, behaviour and toxic reactions. The extracts were safe up to the topical dose of 1000 mg/kg and from results suitable dose was chosen for further activity.
2.3. Preparation of extracts 2.7. Acute oral toxicity [34] The flowers of Woodfordia fructicosa and leaves of Gardenia gummifera were dried under shade in college laboratory. It was pulverized to coarse powder. These powders of plants were used for extraction. The ethanolic extract was prepared by soxhlet extraction method by taking 200 g of powdered and extracting with 500 ml of ethanol for 4 days. Extracts were filtered; filtrates were evaporated at room temperature to dryness. The extracts were used to prepare gel and suspension. 2.4. Formulation development 2.4.1. Gel 1 gm of Carbopol 934 was dispersed in 50 ml of distilled water in a 100 ml. The beaker was kept aside to let the carbopol swell for half an hour and then constant steering (300 RPM for 30 min) was done to mix the carbopol. Required quantity of methyl paraben and EDTA was dissolved in 5 ml of distilled water by heating on water bath. Solution was cooled and Propylene glycol 400 was added.
Acute Oral toxicity study was evaluated as per OECD guidelines 425 on Wistar albino rats. Nine animals were divided in three groups, each group comprises three animals. Group I animals were considered as control, Group II and Group III animals received 1000 mg/kg body weight (limit test) of ethanolic extract of Woodfordia fructicosa flowers and Gardenia gummifera leaves respectively by gavage using oral canula. The extracts were safe up to the oral dose of 1000 mg/kg and from results suitable dose was chosen for further activity. 2.8. Evaluation of anti-psoriatic activity [35] 2.8.1. Preliminary induction of psoriasis Eight Wistar rats weighing around 120 g were used & divided into four groups each comprises two rats. Hairs on the dorsal skin were removed using hair removing cream (Veet). A 10% body surface area was irradiated for 15, 30 and 45 min at a vertical
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distance of 20 cm with UV light (385 nm) in group 2, 3 & 4 respectively. Group 1 was considered as control (No radiation). After irradiation, rats were monitored for observing any change on the irradiated skin, any appearance of skin lesion and other different behaviours. 2.8.2. Anti-psoriatic activity of topical formulations A total 30 Wistar rats weighing around 120–140 g were used. Psoriasis was induced in animals as mentioned in preliminary induction protocol. After induction, animals were divided into five groups comprising of 5 animals in each group as follows. Group I: Normal control (Vehicle treated & no induction) Group II: Disease control (psoriasis induction but untreated) Group III: Induced psoriasis Treated with 0.05% gel of Tretinoin Microsphere gel 0.04% w/w (Supatret 0.04) once daily for 12 days. Group IV: Induced psoriasis Treated with 0.1% gel of extract of Woodfordia fructicosa once daily for 12 days. Group V: Induced psoriasis Treated with 0.1% gel of extract of Gardenia gummifera once daily for 12 days.
for a period of 4 h. The hydroxyproline content in each sample analyzed was found from a calibration curve. 2.12. Statistical analysis All the values are expressed as mean standard error of mean (S.E.M.) and analyzed for ANOVA and posthoc Tukey-Kramer Multiple Comparisons Test by employing statistical software, GraphPad Prism 7. 3. Results 3.1. Acute dermal toxicity The extracts were safe up to the dose of 1000 mg/kg. There were no changes in fur, eyes, and behaviour of treated animals as well as no toxic reactions determined and from results suitable dose (0.1% w/w) was chosen for each gel for further studies. 3.2. Acute oral toxicity
2.8.3. Anti-psoriatic activity of oral formulations In the experiment, a total of 20 rats were used. The rats were divided into 4 groups comprising of 5 animals in each group as follows: Group I: Normal control (Vehicle treated & no induction) Group II: Disease control (psoriasis induction but untreated) Group III: Animals Treated with suspension of extract of Woodfordia fructicosa (100 mg/kg body wt.) once daily for 12 days. Group IV: Animals Treated with suspension of extract of Gardenia gummifera (100 mg/kg body wt.) once daily for 12 days.
Animals were observed individually for any toxicity sign of gross changes like convulsion, tremor, circling, depression, and mortality after dosing for 24 h with special attention given during the first 4 h, and thereafter, 24 h. All observations were systematically recorded with individual records being maintained for each animal. Administered dose was found tolerable (no death found) and from results suitable dose (100 mg/kg body wt.) was chosen for each suspension for further studies. 3.3. Macroscopic examination
2.9. Macroscopic examination Animals were evaluated by severity index (severity score) of psoriatic lesions every third day. A visual (by naked eyes) scoring system was developed based on Severity Index (SI). SI was scored on a scale from 0 to 3: 0- none (clear); 1- mild (redness); 2moderate (redness and erythema); 3- sever (redness, erythema and scaling). 2.10. Histopathological examination At the end of study, animals were anaesthetized using ketamine. Specimens of skin (tissues) were collected & preserved in glass vials containing 10% formalin solution. Longitudinal sections of skin specimen (about 5 mm thickness) were prepared by microtomy and stained with hematoxylin-eosin dye for histological examination. The thickness of the cellular part of epidermis was determined using a calibrated ocular micrometer and all measurements were adjusted for magnification optics. 2.11. Biochemical estimation [36] For the estimation of hydroxyproline content, the tissues were excised and store in formaline solution at 20 C until used. Tissue samples were hydrolyzed in parex tubes with 6N HCl for 3 h at 130 C. The hydrolysate was neutralized to pH 7.0 and was subjected to Chloramine-T oxidation for 20 min. Five min after addition of oxidizer, 2.5 ml each of Ehrlich reagent was poured into every test tube, and they were immersed in a water bath at 60 C. After 25 min, the test tubes were transferred to an ice bath and, after cooling, 6.6 ml each of isopropyl alcohol was added. After thorough stirring, the samples were analyzed in the spectrophotometer, in 1 cm cuvettes, at a wavelength of 557 nm, against a control, in which the solution being analyzed was replaced by water. The colour developed practically does not change intensity
3.3.1. Preliminary induction of psoriasis Immediately after irradiation, initial faint erythema appears and disappears within 30 min. The second phase of erythema starts 24 h after the irradiation and gradually increases. 5 day after irradiation, macroscopic observations were analysed and found maximum severity score in group 4 animals. Results was observed in all groups and found better psoriatic lesions (maximum severity index) in 45 min irradiated rats. Finally 45 min duration of exposure was considered for induction in final protocol. The results are shown in Table 1 & Fig. 1. 3.3.2. Topical formulation The results of the topical formulations on severity index are given in Table 2. Severity Index was evaluated by observing the psoriatic lesions by naked eyes in each 4 days interval. Results reveal that both test groups (IV & V) and standard group showed day to day decreases severity index. On day 4, standard group treated 23.07% of psoriatic lesion while test group IV treated 15.38% and group V showed no effect as compared to disease control group (G-II). On day 8, both standard group and test group Table 1 Macroscopic observations in preliminary induction of psoriasis in rats. Groups
Animal
I
1 2 1 2 1 2 1 2
II III IV
Severity Index Day 1
Mean
Day 3
Mean
Day 5
Mean
0 0 0 0 0 0 1 1
0
0 0 0 1 2 2 2 2
0
0 0 1 1 3 2 3 3
0
0 0 1
0.5 2 2
1 2.5 3
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Fig. 1. Preliminary induction of psoriasis by UV Radiation in rats.
Table 2 Effect of topical formulations on macroscopic features in UV Radiation induced psoriasis in rats. Groups Severity Index
I II III IV V
Day 4
% Decrease
Day 8
% Decrease
Day 12
% Decrease
0 0.0 2.6 0.24 2.0 0.0 2.2 0.2 2.6 0.24
– – 23.07 15.38 0.0
0 0.0 1.6 0.24 1.2 0.2 1.2 0.2 1.4 0.24
– – 25.0 25.0 12.5
0 0.0 0.6 0.24 0 0.0 0.2 0.2 0.4 0.24
– – 100 66.66 33.33
IV treated 25% of psoriatic lesion while test group V treated 12.5%. On day 12, standard group completely treated (100%) of psoriatic lesion while test group IV treated 66.6% and test group V treated 33.33%. 3.3.3. Oral formulation The results of the oral formulations on severity index are given in Table 3. Results reveal that both test group (III & IV) showed day to day decreases severity index. On day 4, test group III treated 21.42% of psoriatic lesion and test group IV treated 14.28%. On day 8, test group III treated 33.33% and test group IV treated 22.22%. On day 12, test group III treated 50% of and test group IV treated 25%.
Severity Index values are expressed as mean SEM (n = 5).
Table 3 Effect of oral formulations on macroscopic features in UV Radiation induced psoriasis in rats. Groups
I II III IV
Severity Index Day 4
% Decrease
Day 8
% Decrease
Day 12
% Decrease
0 0.0 2.8 0.2 2.2 0.2 2.4 0.2 4
– – 21.42 14.28
0 0.0 1.8 0.2 1.2 0.2 1.4 0.24
– – 33.33 22.22
0 0.0 0.8 0.2 0.4 0.24 0.6 0.24
– – 50.0 25.0
Severity Index values are expressed as mean SEM (n = 5).
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3.4. Epidermal thickness 3.4.1. Topical formulation The results of the topical formulations on epidermal thickness are given in Fig. 2. The microscopic evaluation revealed significant reduction (P < 0.001) in epidermal thickness. The epidermal thickness of the test group IV& V was found to be almost equal 31.2 0.46 mm & 31.3 0.37 mm respectively which was lesser than that of disease control group 45.9 0.33 mm and slightly higher than that of standard group 30.7 0.51 mm, which indicate good anti-psoriatic strength of the topical formulations of both extracts. 3.4.2. Oral formulation The results of the oral formulations on epidermal thickness are given in Fig. 3. The microscopic evaluation revealed significant reduction (P < 0.001) in epidermal thickness. The epidermal thickness of the test group III & IV was found to be 38.6 0.43 mm & 44.2 0.25 mm respectively which was lesser than that of disease control group 46.5 0.22 mm, which indicate good anti-psoriatic strength of the oral formulations of both extracts.
Fig. 4. Effect of topical formulations on hydroxyproline content in UV Radiation induced psoriasis in rats. All values are mean SEM, n = 5.
3.5.2. Oral formulation The results of the oral formulations on hydroxyproline content are given in Fig. 5. The biochemical evaluation revealed no significant reduction in hydroxyproline content. The hydroxyproline content of the test group III & IV was found to be 17.87 1.72 &15.19 2.08 respectively which was higher than that of disease control group 14.5 1.46 mg/100 mg tissues.
3.5. Hydroxyproline content 3.6. Histopathological examination 3.5.1. Topical formulation The results of the topical formulations on hydroxyproline content are given in Fig. 4. The biochemical evaluation revealed no significant reduction in hydroxyproline content. The hydroxyproline content of the test group IV& V was found to be almost equal 14.98 2.24& 14.84 1.1 respectively which was slightly higher than that of disease control group 14.79 2.81 and lesser than that of standard group 17.5 2.52 mg/100 mg tissues.
Fig. 2. Effect of topical formulations on Epidermal Thickness in UV Radiation induced psoriasis in rats. All values are mean SEM, n = 5, **p < 0.01, ***p < 0.001.
Fig. 3. Effect of oral formulations on Epidermal Thickness in UV Radiation induced psoriasis in rats. All values are mean SEM, n = 5, ***p < 0.001.
Sections were also examined the presence of Histopathological features (elongation of rete ridges and capillary loop dilation) by direct microscopy which were also support the macroscopic and biochemical studies. The results are shown in Fig. 6. 4. Discussion Skin of all types can be damaged by UV Radiation (UVR) but lighter skin is more sensitive than heavily pigmented skin. The effect of UV Radiation on the skin is quantified by the Minimal Erythemal Dose (MED). The MED is the lowest dose of UV Radiation that will produce a barely detectable erythema (reddening of the skin). Its actual value depends on skin type and on the definition of minimal erythema. Lightly pigmented skin is more susceptible to the effects of UVR [37]. Exposure to UV Radiation, particularly from wavelengths less than 315 nm, can result in erythema or sunburn. The redness of the skin which is characteristic of erythema is attributable to increased blood content by dilation of the superficial blood vessels, mainly the sub papillary venules [38]. The papillary capillaries themselves add little to the redness seen [39]. Natural sunburn has latent period of a few hours and once present may persist for several hour or even a few days. Erythema induced by artificial sources is strongly dependent on the wavelength of radiation. At 300 nm an average threshold dose in
Fig. 5. Effect of oral formulations on hydroxyproline content in UV Radiation induced psoriasis in rats. All values are mean SEM, n = 5.
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Fig. 6. Effect of topical and oral formulations on histopathological features in UV Radiation induced psoriasis in rats.
white skin lies at about 10–20 mJ cm 2 [40]. The vascular response due to UVR could be considered to arise from two different types of mechanism. It could be from a direct action on the vessel wall itself, or indirectly from a photochemical reaction via a diffusing chemical mediator arising in the epidermis [39]. In a comment on ultraviolet erythemas in man, a researcher has examined the evidence that prostaglandins act as mediators or modulators of inflammation in UVR erythema. Researcher postulates that uvc erythema is closely associated with the formation of prostaglandins as a result of damage to the epidermis, which then diffuse into the dermis and cause vasodilation [41]. Psoriasis is a common genetically determined chronic inflammatory skin disorder characterized by red, scaly and raised patches. In psoriasis, epidermal hyperproliferation, abnormal keratinocyte differentiation, angiogenesis with blood vessel dilatation and excess Th-1 and Th-17 inflammation can be observed [42]. However, although genetic, immunological and environmental factors seem implied, the exact cause is not yet known and even today, psoriasis is not well understood [43]. Histological appearance of lesions shows elongation of the rete ridges, disappearance of the granular layer and persistence of the keratinocytes nuclei in the stratum corneum of the epidermis [44]. An increase in collagen catabolism and regeneration is an important link in the pathogenesis of many diseases. Twelvefourteen percent of all amino acid residues of collagen are hydroxyproline [45]. Collagen is a major constituent of the dermis, and is therefore involved in many facets of skin disease (including psoriasis) as well as the recovery process. Collagen also plays an important role in maintaining the moisture content and the elasticity of skin, although collagen is altered and damaged by aging and/or ultraviolet rays [46,47]. Generally, in order to avoid such damage and changes, attempts have been made to regenerate collagen in skin by stimulation with either a laser or far-Infra Red rays [48]. However, these treatments induce erythema or redness of the skin for a few weeks to several months [49,50]. A broad spectrum of anti-psoriatic treatments, both topical and systemic, is available for the management of psoriasis [51]. However, it is often resistant to treatment or, else, frequently
relapses upon cessation of medication after partial or acceptable clearance is obtained [52]. The severity of the disease usually determines the therapeutic approach. Among the treatments, there are topical (Corticosteroids, Vitamin D analogues, Tazarotene, Calcineurin inhibitors), phototherapy (Narrowband UVB, Broadband UVB, Psoralen-UVA, Excimer laser), biologic (Alefacept, Efalizumab, Etanercept, Infliximab, Adalimumab) and systemic treatments (Cyclosporine A, Methotrexate, Acitretin, Fumaric acid esters, Sulfasalazine, Mycophenolate mofetil, 6-Thioguanine, Hydroxyurea). Approximately, 70 to 80% of all patients with psoriasis can be treated adequately with topical therapy [53]. For others, phototherapy and systemic treatments are effective; however, the duration of a treatment is restricted because of the cumulative toxicity potential of an individual therapy [52]. For example, some treatments may increase the risk of cancer (phototherapy) while others can induce disorders in the liver (Methotrexate) [54]. Sometimes, treatment efficacy may diminish with time and it must be replaced by another therapy [51]. At the present time, there is still no curative treatment for psoriasis. 5. Conclusion Prepared formulations (topical as well as oral) of ethanolic extract of Woodfordia fructicosa flowers and Gardenia gummifera leaves decreased severity index, epidermal thickness as well as other histopathological features and also increased hydroxyproline content. From the preliminary in vivo study we concluded that extractive Phytoconstituents of Woodfordia fructicosa flowers and Gardenia gummifera leaves have potent anti-psoriatic activity. Further research is warranted to elucidate the specific phytoconstituent/s involved. Acknowledgement Authors are thankful to Truba Institute of Pharmacy and Sapience Bioanalytical Research Lab. Bhopal, India for providing necessary research facilities for the successful completion of research work.
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Original article
Evaluation of potent phytomedicine for treatment of psoriasis using UV radiation induced psoriasis in rats Hemant K. Nagara,* , Amit K. Srivastavab , Rajnish Srivastavac, Mahendra S. Ranawata a
Bhupal Nobles' College of Pharmacy, Udaipur 313002, India Indian Institute of Integrative Medicine, (Council of Scientific & Industrial Research), Jammu- 180001, India c Moradabad Educational Trust, Group of Institutions, Faculty of Pharmacy, Moradabad 244001, India b
A R T I C L E I N F O
Article history: Received 10 August 2016 Received in revised form 8 October 2016 Accepted 9 October 2016 Keywords: UV radiation Psoriasis Woodfordia fructicosa Gardenia gummifera Severity index Epidermal thickness Hydroxyproline content
A B S T R A C T
The aim of present study was to determine the effect of newly formulated gels and suspensions of extractive Phytoconstituents of Woodfordia fructicosa flowers and Gardenia gummifera leaves by using UV Radiation induced psoriasis in rats. Both plants are traditionally claimed to be useful in treatment of number of skin diseases. However, there are no established scientific reports for their potential in psoriasis. Formulated Gels and Suspensions of ethanolic extract of both plants were tested for acute dermal and oral toxicity study respectively. The results of acute dermal toxicity at concentration 1% w/w and oral toxicity at dose 1000 mg/kg showed that the gels and suspensions were safe. Psoriasis was induced in Wistar rats by espousing 10% area of total body by UV radiations. Anti-psoriatic activity was performed by applying 0.1% gel and orally at a dose 100 mg/kg body weight in rats. Severity Index, histological study and biochemical estimation were analyzed. The results of our studies showed that the test formulations (Gels and Suspensions) of both plant extracts exhibited potential effect in anti-psoriatic activity. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Many herbal remedies individually or in combination have been recommended in various medical expositions for the cure of different diseases. Plants are among the most important and common sources of potentially valuable new drugs. Therefore, there is a need to investigate the biological properties of medicinal plants in order to develop new drugs. Much work has been carried out on herbal treatment of various diseases, but still there is need to explore more. Herbal remedies are promising in the management of dermatological conditions including psoriasis [1]. Psoriasis is a common skin disease affecting 2% of world population characterized by epidermal keratinocyte hyper proliferation, abnormal keratinocyte differentiation and immune cell infiltration [2–4]. Psoriasis is a recurrent and debilitating disease. The existing topical treatments such as emollients, coal tar and dithranol had lower efficacy and cosmetically unacceptable, while systemic therapies such as methotrexate, cyclosporine and acitretin had significant side effects [5]. Overexposure to ultraviolet (UV) radiation can cause a number of skin disorders such as erythema,
* Corresponding author. E-mail address: [email protected] (H.K. Nagar). http://dx.doi.org/10.1016/j.biopha.2016.10.027 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
edema, hyperpigmentation, immunosuppression, photoaging and skin cancer. Since the level of UV radiation is increasing as a result of depletion of the stratospheric ozone and climate change, the protection of human skin against the harmful effects of UV radiation is an urgent need [6]. Woodfordia fructicosa (Kurz.) family Lythraceae is a straggling leafy shrub, frequently used English names are Fire Flame Bush while locally known as Dhai is about 3.5 m in high and distributed abundantly throughout the North India, as well as in the majority of the East Asian countries. According to the Traditional system of medicine, the flowers of Woodfordia fructicosa are pungent, acrid, cooling, toxic, alexiteric, use as astringenttonic in disorders of mucous membranes, uterine sedative and anthelmintic and is useful in thirst, dysentery, leprosy, erysipelas, blood disease, leucorrhoea, menorrhagia and toothache [7,8]. In addition, Woodfordia fructicosa flowers are extensively used by tribal people for its wound, ulcer healing, analgesic and anti-rheumatic properties [9– 11]. Pharmacological claims of Woodfordia fructicosa dried flowers can be ascribed for its important bioactive phytoconstituents such as flavonoids, sterols, anthraquinones, saponins and tannins [12– 14]. Preclinical data from various studies indicates, that dried Woodfordia fructicosa flower extract possess anti-pyretic, Antiinflammatory [15], Anti-tumor [16,17], Anti-viral [18], Immunomodulatory [19], Anti-fertility [20], Antibacterial [21],
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Hepatoprotective [22], Anti-hyperlipedemic [23], Antidiabetic [24], Bronchoprotective [25], Wound healing [26] and Antiasthmatic activity [27]. Gardenia gummifera (family- Rubiaceae) is geographically distributed in India, Burma, Bangladesh, Konkan region, North Kanara, and Malabar Coast. G. gummifera is claimed to have a number of medicinal properties which include anthelmintic, antispasmodic, carminative, diaphoretic, expectorant, potentiation of pentobarbitone induced sleep, antiepileptic, peripheral and central analgesic, cardiotonic, antioxidant and anti-hyperlipidemic. It is also claimed to be useful in dyspepsia, flatulence for cleaning foul ulcers and wounds and to keep off flies from wounds in veterinary practice [28,29]. Dikamali is the gum resin obtained from the leaf buds of Gardenia gummifera [30]. A number of flavonoids such as gardenin A, B, C, D and E were isolated from the plant [31,32]. The close resemblance of inflammatory process produced by ultraviolet radiation to the one exhibited in psoriasis provides us with a good model to investigate drugs that have a potential to reduce the inflammatory reaction associated with psoriasis. Hence, Anti-psoriatic activity of phytomedicine extracted from Woodfordia fructicosa and Gardenia gummifera is evaluated using UV Radiation induced psoriasis in experimental rats. 2. Materials and methods 2.1. Chemicals Tretinoin Microsphere gel 0.04% w/w (Supatret 0.04) manufectured by sun pharmaceuticals Pvt. Ltd was purchased from medical store. Chloramine-T and Ehrlich reagent were purchased from Himedia laboratories Pvt. Ltd for hydroxyproline estimation. All other chemicals used for this study were of analytical grade. 2.2. Plant materials The flowers of Woodfordia fructicosa and leaves of Gardenia gummifera Linn were collected in the month of February 2014 from the Swarn Jayanti Park, near Sarvadharm colony, Bhopal (M.P.). Both the plant were authenticated by Dr. Zia Ul Hasan (Professor, Department of Botany), Safia College Bhopal. The voucher specimen for W. Fructicosa (460/Bot/Saif/14) and G. Gummifera (503/Bot/Saif/14) has been deposited in the herbarium section of Department of Botany, Safia College, Bhopal for future and further reference.
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Further required quantity of extract (100 mg for 0.1%) was mixed to the above mixture and volume made up to 100 ml by adding remaining distilled water. Finally full mixed ingredients were mixed properly to the Carbopol gel with continuous stirring and triethanolamine was added drop wise to the formulation for adjustment of required skin pH and to obtain the gel at required consistency. 2.4.2. Suspension Suspensions of extracts were prepared using 0.5% carboxyl methyl cellulose (CMC) as suspending agent. Calculated amount of extract was premixed in distilled water followed by addition of required amount of CMC, Sorbitol Solution (0.5%) and Methyl Paraben; the mixture was agitated with the help of glass rod and was finally sonicated with help of ultrasonicator. Suspensions of both extract were prepared to get the test doses (50 mg/kg per ml). 2.5. Animals care and acclimatization The animal experimental protocol was approved by the Institutional Animals Ethical Committee (IAEC), Sapience Bioanalytical Research Lab. (Reg. no. 1413/PO/E/S/11/CPCSEA), Bhopal, India. Protocol Approval Number is SBRL/IAEC/June 2014/14. Male & female Wistar albino rats (120–140 g) were provided by Sapience Bioanalytical Research Lab. The animals were housed in standard conditions of temperature (25 2 C) and 12:12 h light-dark cycle. The rats were fed with commercial diet and water ad Libitum. The animals were acclimatized to the laboratory conditions for a minimum period of seven days prior to commencement of treatment. 2.6. Acute dermal toxicity [33] The acute dermal toxicity test of extracts was determined according to the OECD guidelines no. 402. Adult Wistar rats of either sex were used. Nine animals were divided in three groups, each group comprises three animals. Approximately 24 h before the test, 10% hairs of the body were removed from the dorsal area of the test animals by using hair removal cream (Veet). Group I animals were considered as control, Group II and Group III animals received topically 1000 mg/kg body weight (limit test) of ethanolic extract of Woodfordia fructicosa flowers and Gardenia gummifera leaves respectively. All animals were monitored for 14 days for changes in fur, eyes, behaviour and toxic reactions. The extracts were safe up to the topical dose of 1000 mg/kg and from results suitable dose was chosen for further activity.
2.3. Preparation of extracts 2.7. Acute oral toxicity [34] The flowers of Woodfordia fructicosa and leaves of Gardenia gummifera were dried under shade in college laboratory. It was pulverized to coarse powder. These powders of plants were used for extraction. The ethanolic extract was prepared by soxhlet extraction method by taking 200 g of powdered and extracting with 500 ml of ethanol for 4 days. Extracts were filtered; filtrates were evaporated at room temperature to dryness. The extracts were used to prepare gel and suspension. 2.4. Formulation development 2.4.1. Gel 1 gm of Carbopol 934 was dispersed in 50 ml of distilled water in a 100 ml. The beaker was kept aside to let the carbopol swell for half an hour and then constant steering (300 RPM for 30 min) was done to mix the carbopol. Required quantity of methyl paraben and EDTA was dissolved in 5 ml of distilled water by heating on water bath. Solution was cooled and Propylene glycol 400 was added.
Acute Oral toxicity study was evaluated as per OECD guidelines 425 on Wistar albino rats. Nine animals were divided in three groups, each group comprises three animals. Group I animals were considered as control, Group II and Group III animals received 1000 mg/kg body weight (limit test) of ethanolic extract of Woodfordia fructicosa flowers and Gardenia gummifera leaves respectively by gavage using oral canula. The extracts were safe up to the oral dose of 1000 mg/kg and from results suitable dose was chosen for further activity. 2.8. Evaluation of anti-psoriatic activity [35] 2.8.1. Preliminary induction of psoriasis Eight Wistar rats weighing around 120 g were used & divided into four groups each comprises two rats. Hairs on the dorsal skin were removed using hair removing cream (Veet). A 10% body surface area was irradiated for 15, 30 and 45 min at a vertical
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distance of 20 cm with UV light (385 nm) in group 2, 3 & 4 respectively. Group 1 was considered as control (No radiation). After irradiation, rats were monitored for observing any change on the irradiated skin, any appearance of skin lesion and other different behaviours. 2.8.2. Anti-psoriatic activity of topical formulations A total 30 Wistar rats weighing around 120–140 g were used. Psoriasis was induced in animals as mentioned in preliminary induction protocol. After induction, animals were divided into five groups comprising of 5 animals in each group as follows. Group I: Normal control (Vehicle treated & no induction) Group II: Disease control (psoriasis induction but untreated) Group III: Induced psoriasis Treated with 0.05% gel of Tretinoin Microsphere gel 0.04% w/w (Supatret 0.04) once daily for 12 days. Group IV: Induced psoriasis Treated with 0.1% gel of extract of Woodfordia fructicosa once daily for 12 days. Group V: Induced psoriasis Treated with 0.1% gel of extract of Gardenia gummifera once daily for 12 days.
for a period of 4 h. The hydroxyproline content in each sample analyzed was found from a calibration curve. 2.12. Statistical analysis All the values are expressed as mean standard error of mean (S.E.M.) and analyzed for ANOVA and posthoc Tukey-Kramer Multiple Comparisons Test by employing statistical software, GraphPad Prism 7. 3. Results 3.1. Acute dermal toxicity The extracts were safe up to the dose of 1000 mg/kg. There were no changes in fur, eyes, and behaviour of treated animals as well as no toxic reactions determined and from results suitable dose (0.1% w/w) was chosen for each gel for further studies. 3.2. Acute oral toxicity
2.8.3. Anti-psoriatic activity of oral formulations In the experiment, a total of 20 rats were used. The rats were divided into 4 groups comprising of 5 animals in each group as follows: Group I: Normal control (Vehicle treated & no induction) Group II: Disease control (psoriasis induction but untreated) Group III: Animals Treated with suspension of extract of Woodfordia fructicosa (100 mg/kg body wt.) once daily for 12 days. Group IV: Animals Treated with suspension of extract of Gardenia gummifera (100 mg/kg body wt.) once daily for 12 days.
Animals were observed individually for any toxicity sign of gross changes like convulsion, tremor, circling, depression, and mortality after dosing for 24 h with special attention given during the first 4 h, and thereafter, 24 h. All observations were systematically recorded with individual records being maintained for each animal. Administered dose was found tolerable (no death found) and from results suitable dose (100 mg/kg body wt.) was chosen for each suspension for further studies. 3.3. Macroscopic examination
2.9. Macroscopic examination Animals were evaluated by severity index (severity score) of psoriatic lesions every third day. A visual (by naked eyes) scoring system was developed based on Severity Index (SI). SI was scored on a scale from 0 to 3: 0- none (clear); 1- mild (redness); 2moderate (redness and erythema); 3- sever (redness, erythema and scaling). 2.10. Histopathological examination At the end of study, animals were anaesthetized using ketamine. Specimens of skin (tissues) were collected & preserved in glass vials containing 10% formalin solution. Longitudinal sections of skin specimen (about 5 mm thickness) were prepared by microtomy and stained with hematoxylin-eosin dye for histological examination. The thickness of the cellular part of epidermis was determined using a calibrated ocular micrometer and all measurements were adjusted for magnification optics. 2.11. Biochemical estimation [36] For the estimation of hydroxyproline content, the tissues were excised and store in formaline solution at 20 C until used. Tissue samples were hydrolyzed in parex tubes with 6N HCl for 3 h at 130 C. The hydrolysate was neutralized to pH 7.0 and was subjected to Chloramine-T oxidation for 20 min. Five min after addition of oxidizer, 2.5 ml each of Ehrlich reagent was poured into every test tube, and they were immersed in a water bath at 60 C. After 25 min, the test tubes were transferred to an ice bath and, after cooling, 6.6 ml each of isopropyl alcohol was added. After thorough stirring, the samples were analyzed in the spectrophotometer, in 1 cm cuvettes, at a wavelength of 557 nm, against a control, in which the solution being analyzed was replaced by water. The colour developed practically does not change intensity
3.3.1. Preliminary induction of psoriasis Immediately after irradiation, initial faint erythema appears and disappears within 30 min. The second phase of erythema starts 24 h after the irradiation and gradually increases. 5 day after irradiation, macroscopic observations were analysed and found maximum severity score in group 4 animals. Results was observed in all groups and found better psoriatic lesions (maximum severity index) in 45 min irradiated rats. Finally 45 min duration of exposure was considered for induction in final protocol. The results are shown in Table 1 & Fig. 1. 3.3.2. Topical formulation The results of the topical formulations on severity index are given in Table 2. Severity Index was evaluated by observing the psoriatic lesions by naked eyes in each 4 days interval. Results reveal that both test groups (IV & V) and standard group showed day to day decreases severity index. On day 4, standard group treated 23.07% of psoriatic lesion while test group IV treated 15.38% and group V showed no effect as compared to disease control group (G-II). On day 8, both standard group and test group Table 1 Macroscopic observations in preliminary induction of psoriasis in rats. Groups
Animal
I
1 2 1 2 1 2 1 2
II III IV
Severity Index Day 1
Mean
Day 3
Mean
Day 5
Mean
0 0 0 0 0 0 1 1
0
0 0 0 1 2 2 2 2
0
0 0 1 1 3 2 3 3
0
0 0 1
0.5 2 2
1 2.5 3
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Fig. 1. Preliminary induction of psoriasis by UV Radiation in rats.
Table 2 Effect of topical formulations on macroscopic features in UV Radiation induced psoriasis in rats. Groups Severity Index
I II III IV V
Day 4
% Decrease
Day 8
% Decrease
Day 12
% Decrease
0 0.0 2.6 0.24 2.0 0.0 2.2 0.2 2.6 0.24
– – 23.07 15.38 0.0
0 0.0 1.6 0.24 1.2 0.2 1.2 0.2 1.4 0.24
– – 25.0 25.0 12.5
0 0.0 0.6 0.24 0 0.0 0.2 0.2 0.4 0.24
– – 100 66.66 33.33
IV treated 25% of psoriatic lesion while test group V treated 12.5%. On day 12, standard group completely treated (100%) of psoriatic lesion while test group IV treated 66.6% and test group V treated 33.33%. 3.3.3. Oral formulation The results of the oral formulations on severity index are given in Table 3. Results reveal that both test group (III & IV) showed day to day decreases severity index. On day 4, test group III treated 21.42% of psoriatic lesion and test group IV treated 14.28%. On day 8, test group III treated 33.33% and test group IV treated 22.22%. On day 12, test group III treated 50% of and test group IV treated 25%.
Severity Index values are expressed as mean SEM (n = 5).
Table 3 Effect of oral formulations on macroscopic features in UV Radiation induced psoriasis in rats. Groups
I II III IV
Severity Index Day 4
% Decrease
Day 8
% Decrease
Day 12
% Decrease
0 0.0 2.8 0.2 2.2 0.2 2.4 0.2 4
– – 21.42 14.28
0 0.0 1.8 0.2 1.2 0.2 1.4 0.24
– – 33.33 22.22
0 0.0 0.8 0.2 0.4 0.24 0.6 0.24
– – 50.0 25.0
Severity Index values are expressed as mean SEM (n = 5).
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3.4. Epidermal thickness 3.4.1. Topical formulation The results of the topical formulations on epidermal thickness are given in Fig. 2. The microscopic evaluation revealed significant reduction (P < 0.001) in epidermal thickness. The epidermal thickness of the test group IV& V was found to be almost equal 31.2 0.46 mm & 31.3 0.37 mm respectively which was lesser than that of disease control group 45.9 0.33 mm and slightly higher than that of standard group 30.7 0.51 mm, which indicate good anti-psoriatic strength of the topical formulations of both extracts. 3.4.2. Oral formulation The results of the oral formulations on epidermal thickness are given in Fig. 3. The microscopic evaluation revealed significant reduction (P < 0.001) in epidermal thickness. The epidermal thickness of the test group III & IV was found to be 38.6 0.43 mm & 44.2 0.25 mm respectively which was lesser than that of disease control group 46.5 0.22 mm, which indicate good anti-psoriatic strength of the oral formulations of both extracts.
Fig. 4. Effect of topical formulations on hydroxyproline content in UV Radiation induced psoriasis in rats. All values are mean SEM, n = 5.
3.5.2. Oral formulation The results of the oral formulations on hydroxyproline content are given in Fig. 5. The biochemical evaluation revealed no significant reduction in hydroxyproline content. The hydroxyproline content of the test group III & IV was found to be 17.87 1.72 &15.19 2.08 respectively which was higher than that of disease control group 14.5 1.46 mg/100 mg tissues.
3.5. Hydroxyproline content 3.6. Histopathological examination 3.5.1. Topical formulation The results of the topical formulations on hydroxyproline content are given in Fig. 4. The biochemical evaluation revealed no significant reduction in hydroxyproline content. The hydroxyproline content of the test group IV& V was found to be almost equal 14.98 2.24& 14.84 1.1 respectively which was slightly higher than that of disease control group 14.79 2.81 and lesser than that of standard group 17.5 2.52 mg/100 mg tissues.
Fig. 2. Effect of topical formulations on Epidermal Thickness in UV Radiation induced psoriasis in rats. All values are mean SEM, n = 5, **p < 0.01, ***p < 0.001.
Fig. 3. Effect of oral formulations on Epidermal Thickness in UV Radiation induced psoriasis in rats. All values are mean SEM, n = 5, ***p < 0.001.
Sections were also examined the presence of Histopathological features (elongation of rete ridges and capillary loop dilation) by direct microscopy which were also support the macroscopic and biochemical studies. The results are shown in Fig. 6. 4. Discussion Skin of all types can be damaged by UV Radiation (UVR) but lighter skin is more sensitive than heavily pigmented skin. The effect of UV Radiation on the skin is quantified by the Minimal Erythemal Dose (MED). The MED is the lowest dose of UV Radiation that will produce a barely detectable erythema (reddening of the skin). Its actual value depends on skin type and on the definition of minimal erythema. Lightly pigmented skin is more susceptible to the effects of UVR [37]. Exposure to UV Radiation, particularly from wavelengths less than 315 nm, can result in erythema or sunburn. The redness of the skin which is characteristic of erythema is attributable to increased blood content by dilation of the superficial blood vessels, mainly the sub papillary venules [38]. The papillary capillaries themselves add little to the redness seen [39]. Natural sunburn has latent period of a few hours and once present may persist for several hour or even a few days. Erythema induced by artificial sources is strongly dependent on the wavelength of radiation. At 300 nm an average threshold dose in
Fig. 5. Effect of oral formulations on hydroxyproline content in UV Radiation induced psoriasis in rats. All values are mean SEM, n = 5.
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Fig. 6. Effect of topical and oral formulations on histopathological features in UV Radiation induced psoriasis in rats.
white skin lies at about 10–20 mJ cm 2 [40]. The vascular response due to UVR could be considered to arise from two different types of mechanism. It could be from a direct action on the vessel wall itself, or indirectly from a photochemical reaction via a diffusing chemical mediator arising in the epidermis [39]. In a comment on ultraviolet erythemas in man, a researcher has examined the evidence that prostaglandins act as mediators or modulators of inflammation in UVR erythema. Researcher postulates that uvc erythema is closely associated with the formation of prostaglandins as a result of damage to the epidermis, which then diffuse into the dermis and cause vasodilation [41]. Psoriasis is a common genetically determined chronic inflammatory skin disorder characterized by red, scaly and raised patches. In psoriasis, epidermal hyperproliferation, abnormal keratinocyte differentiation, angiogenesis with blood vessel dilatation and excess Th-1 and Th-17 inflammation can be observed [42]. However, although genetic, immunological and environmental factors seem implied, the exact cause is not yet known and even today, psoriasis is not well understood [43]. Histological appearance of lesions shows elongation of the rete ridges, disappearance of the granular layer and persistence of the keratinocytes nuclei in the stratum corneum of the epidermis [44]. An increase in collagen catabolism and regeneration is an important link in the pathogenesis of many diseases. Twelvefourteen percent of all amino acid residues of collagen are hydroxyproline [45]. Collagen is a major constituent of the dermis, and is therefore involved in many facets of skin disease (including psoriasis) as well as the recovery process. Collagen also plays an important role in maintaining the moisture content and the elasticity of skin, although collagen is altered and damaged by aging and/or ultraviolet rays [46,47]. Generally, in order to avoid such damage and changes, attempts have been made to regenerate collagen in skin by stimulation with either a laser or far-Infra Red rays [48]. However, these treatments induce erythema or redness of the skin for a few weeks to several months [49,50]. A broad spectrum of anti-psoriatic treatments, both topical and systemic, is available for the management of psoriasis [51]. However, it is often resistant to treatment or, else, frequently
relapses upon cessation of medication after partial or acceptable clearance is obtained [52]. The severity of the disease usually determines the therapeutic approach. Among the treatments, there are topical (Corticosteroids, Vitamin D analogues, Tazarotene, Calcineurin inhibitors), phototherapy (Narrowband UVB, Broadband UVB, Psoralen-UVA, Excimer laser), biologic (Alefacept, Efalizumab, Etanercept, Infliximab, Adalimumab) and systemic treatments (Cyclosporine A, Methotrexate, Acitretin, Fumaric acid esters, Sulfasalazine, Mycophenolate mofetil, 6-Thioguanine, Hydroxyurea). Approximately, 70 to 80% of all patients with psoriasis can be treated adequately with topical therapy [53]. For others, phototherapy and systemic treatments are effective; however, the duration of a treatment is restricted because of the cumulative toxicity potential of an individual therapy [52]. For example, some treatments may increase the risk of cancer (phototherapy) while others can induce disorders in the liver (Methotrexate) [54]. Sometimes, treatment efficacy may diminish with time and it must be replaced by another therapy [51]. At the present time, there is still no curative treatment for psoriasis. 5. Conclusion Prepared formulations (topical as well as oral) of ethanolic extract of Woodfordia fructicosa flowers and Gardenia gummifera leaves decreased severity index, epidermal thickness as well as other histopathological features and also increased hydroxyproline content. From the preliminary in vivo study we concluded that extractive Phytoconstituents of Woodfordia fructicosa flowers and Gardenia gummifera leaves have potent anti-psoriatic activity. Further research is warranted to elucidate the specific phytoconstituent/s involved. Acknowledgement Authors are thankful to Truba Institute of Pharmacy and Sapience Bioanalytical Research Lab. Bhopal, India for providing necessary research facilities for the successful completion of research work.
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