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Antidiabetic potential of unripe Carissa carandas Linn. fruit extract
Journal of Ethnopharmacology 135 (2011) 430–433
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Antidiabetic potential of unripe Carissa carandas Linn. fruit extract Prakash R. Itankar ∗ , Sarika J. Lokhande 1 , Prashant R. Verma 2 , Sumit K. Arora 3 , Rajesh A. Sahu 4 , Arun T. Patil 5 Department of Pharmaceutical Sciences, Pharmacognosy and Phytochemistry Division, Rashtrasant Tukadoji Maharaj Nagpur University, Amravati Road, Nagpur 440 033, Maharashtra, India
a r t i c l e
i n f o
Article history: Received 24 October 2010 Received in revised form 10 March 2011 Accepted 14 March 2011 Available online 23 March 2011 Keywords: Antidiabetic Carissa carandas Flavonoid Flavanone Polymerization
a b s t r a c t Ethnopharmacological relevance: Carissa carandas commonly known as Karanda have a long history of use in traditional system of medicine. It is used by tribal healers of Western Ghat region of Karnataka as hepatoprotective and antihyperglycemic. However, no scientific data is available to validate the folklore claim. The present study has been designed to evaluate its unripe fruit for the antidiabetic activity. Aim: In the present study, methanol extract of unripe fruits and its fractions were studied for its antidiabetic potential. Materials and methods: The methanol extract and its fractions were screened for antidiabetic activity in alloxan induced diabetic rats. The polyphenolic, flavonoid and flavanone contents of methanolic extract and its fractions were also determined and correlated with its antidiabetic activity. Results: The experimental data indicated that the methanol extract and its ethyl acetate soluble fraction has significantly lowered the elevated blood glucose levels by 48% (p < 0.001) and 64.5% (p < 0.001) respectively at dose level of 400 mg/kg per oral after 24 h as compared to diabetic control. In order to assess the role of polyphenolic components in the relevant activity, polyphenolic and flavonoid contents were determined. The polyphenolic and flavonoid content of methanol extract and its ethyl acetate soluble fraction were found to be 15.8 ± 1.2 mg and 18.55 ± 0.34 mg (gallic acid equivalent/g extract) and flavonoid content 2.92 ± 0.03 mg and 1.534 ± 0.30 mg (rutin equivalent/g extract) respectively. Conclusion: The increased antidiabetic potential of ethyl acetate fraction over methanol extract is due to its partial purification achieved by fractionation which resulted in increase in degree of polymerization and segregation of secondary metabolites. © 2011 Published by Elsevier Ireland Ltd.
1. Introduction Diabetes mellitus (DM) is a major endocrine disorder, affecting approximately 5% of the world’s population. Diabetes is characterized by abnormalities in carbohydrate, lipid and lipoprotein metabolisms, which not only lead to hyperglycaemia but also cause many complications such as hyperlipidemia, hyperinsulinemia, hypertension and atherosclerosis (Luo et al., 2004; Sepici et al., 2004).
∗ Corresponding author. Tel.: +91 712 2550324; fax: +91 712 2500355; mobile: +91 9423677401. E-mail addresses: pri [email protected] (P.R. Itankar), [email protected] (S.J. Lokhande), [email protected] (P.R. Verma), sum [email protected] (S.K. Arora), [email protected] (R.A. Sahu), [email protected] (A.T. Patil). 1 Mobile: +91 8928002537. 2 Mobile: +91 9423100313. 3 Mobile: +91 9960703930. 4 Mobile: +91 9373102633. 5 Mobile: +91 9422808227. 0378-8741/$ – see front matter © 2011 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2011.03.036
Carissa carandas commonly known as Karanda belongs to family Apocynaceae. In recent years its botanical name was changed to Carissa congesta Wight (syn. Carissa carandas Auct. Formerly widely known as Carissa carandas L.) (Anonymous, 1985). Carissa carandas is large dichotomously branched evergreen shrub with short stem and strong thorn in pairs. This species is a rank-growing, straggly, woody, climbing shrub, usually growing to 10 or 15 ft (3–5 m) high, sometimes ascending to the tops of tall trees. The plant is native and common throughout India (Kirtikar and Basu, 2003). Traditionally, whole plant and its parts were used in the treatment of various ailments. The roots were employed as a bitter stomachic, vermifuge and as an ingredient in the remedy for itches. The roots were reported to contain salicylic acid and cardiac glycosides. It also contains carissone; d-glycoside of -sitosterol; glucosides of odoroside H; carindone; a terpenoid lupeol; ursolic acid and its methyl ester; also carinol, a phenolic lignan. Fruits contains good amount of vitamin C. The fruits, leaves and bark are rich in tannins (Morton, 1987). In Ayurveda, the unripe fruits were used as an anthelmintic, astringent, appetizer, antipyretic, antidiabetic, aphrodisiac, in biliary disorders, stomach disorders, rheumatism and diseases of the
P.R. Itankar et al. / Journal of Ethnopharmacology 135 (2011) 430–433
431
Fig. 1. Effect of Carissa carandas fruit extract and fractions on diabetic animals. Each value represents mean ± S.E., n = 5; CCM, methanolic extract; CCES, ethylacetate soluble fraction of methanolic extract; CCAS, acetone soluble fraction of methanol extract; CCAIS, acetone insoluble fraction of methanolic extract. *Represents statistical significance vs. control (p < 0.05). **Represents statistical significance vs. control (p < 0.001).
brains (Morton, 1987; Iyer and Dubhash, 2006). It is useful in treatment of diarrhea, anorexia and intermittent fevers. Fruits have also been studied for its analgesic, anti-inflammatory and lipase 1 activities (Balakrishnan and Bhaskar, 2009). It is used by tribal healers of Western Ghat region of Karnataka as hepatoprotective and antihyperglycemic. However, no scientific data is available to validate the folklore claim (Kirtikar and Basu, 2003; Christophe, 2006). Keeping the above information in view, the present study has been designed to evaluate its antidiabetic activity. 2. Materials and methods 2.1. Animals Swiss albino rats of Sprague–Dawley strain (200–250 g) of either sex obtained from animal house of our institute were used. The animals were fed a standard pellet diet and water ad libitum. They were maintained in a controlled environment and temperature (22 ± 5 ◦ C with 12-h of light/dark cycle). All experimental protocols were approved by the Institutional Animal Ethical Committee (12/2009/CPCSEA). 2.2. Chemicals and standard drugs Aluminum trichloride hexahydrate, glacial acetic acid, anhydrous sodium carbonate, Folin–Ciocalteu phenol reagent, naringin, gallic acid, rutin (hydrate, min 95%), sodium acetate and 2,4dinitrophenylhydrazine were of analytical grade obtained from S. D. Fine Chemicals Pvt. Ltd. (Vadodra, India). 2.3. Plant material, preparation of extract and its fractionation The fruits were collected locally, authenticated by the Department of Botany, R. T. M. Nagpur University Campus, Nagpur. A voucher specimen has been deposited in the Herbarium of Department of Botany, with collection number RA 9496. The fruits were dried under shade and pulverized to a coarse powder. The powdered crude material was defatted with petroleum ether and then extracted with methanol. The methanol extract (CCM) was concentrated in a rotary vacuum evaporator to yield a dark brown mass (yield 28.07%, w/w). For fractionation, methanol extract was triturated with silica (1:3), loaded to Soxhlet assembly and extracted by ethyl acetate to yield ethyl acetate soluble fraction (CCES; yield: 21.65%, w/w) (Fuentes and Alarcón, 2006). The ethyl acetate insoluble portion was further extracted with acetone to yield acetone soluble fraction (CCAS; yield 10.56%,
w/w) and acetone insoluble fraction (CCAIS; yield: 67.19%, w/w). The methanol extract (CCM) and these three broad fractions i.e. CCES, CCAS and CCAIS were subjected to phytochemical and pharmacological screening. 2.4. Phytochemical screening (Stahl, 1969; Harbone, 1976; Wagner et al., 1984) The CCM extract and its broad fractions were screened for the presence of tannins, saponins, unsaturated sterols, triterpenes, alkaloids, flavonoids, lactones/esters, protein/amino acids and carbohydrates and/or glycosides with thin layer chromatography (TLC). Thin layer plates precoated with silica gel G (Merck, 0.25 mm thickness) were used. Development was carried out with different solvent systems such as ethyl acetate:methanol:water (100:13.5:10, v/v/v), ethyl acetate:formic acid:acetic acid:water (100:11:11:26, v/v/v/v), chloroform:methanol:water (70:30:4, v/v/v), toluene:ethyl acetate:diethylamine (70:20:10, v/v/v) and ethyl acetate:methanol:water:acetic acid (65:15:15:10, v/v/v/v). After development of chromatogram in the solvents the plates were dried and sprayed with Dragendorff’s, AlCl3 , hydroxylamine-ferric chloride, ninhydrin and antimony trichloride for the detection of alkaloids, flavonoids, lactones/esters, protein/amino acids, unsaturated sterols and triterpenes respectively. While detection of anthraquinones, saponins, tannins, carbohydrate and/or glycosides is carried out using KOH, anisaldehyde-sulphuric acid reagent, ferric chloride and naphthoresorcinol reagent respectively and visualization is carried out under visible and UV light (: 366 nm). CCM extract and its fractions were also quantified for presence of important secondary metabolites such as total polyphenol, flavonoid and flavonone compounds using following spectroscopic methods. 2.4.1. Determination of total polyphenol compounds (TP), flavonoids (TFA) and total flavanones (TFO) Total polyphenol content was measured using Folin–Ciocalteu colorimetric method (Singleton and Rossi, 1965; Singlenton et al., 1999). Gallic acid was used as a reference for constructing the standard curve (10–100 mg/ml). The results were expressed as mg of gallic acid equivalents (GAE)/g of extract. Flavonoid content was determined by the aluminum chloride method (Jin-Yuarn and Ching-Yin, 2007; Stanojevic´ et al., 2009). While the modified 2,4-dinitrophenylhydrazine method was used for determination of flavanones (Nagy and Grancai, 1996). Rutin and naringin were used as a reference standard and results were
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Table 1 Effect of Carissa carandas fruit extract and fractions on oral glucose tolerance test. Group
Extract/fractions
Blood glucose concentration (mg/dl)
I II III IV V VI VII VIII IX X
Control Metformin (50 mg/kg i.p.) CCM extract (200 mg/kg p.o.) CCM extract (400 mg/kg p.o.) CCES fraction (200 mg/kg p.o.) CCES fraction (400 mg/kg p.o.) CCAS fraction (200 mg/kg p.o.) CCAS fraction (400 mg/kg p.o.) CCAIS fraction (200 mg/kg p.o.) CCAIS fraction (400 mg/kg p.o.)
89 83 85 85 83 82 87 84 87 86
0 min ± ± ± ± ± ± ± ± ± ±
1.7* 1** 3.1* 3* 1.9 2.1** 3.1 2.2** 3.1** 2.2*
30 min 113 99 102 101 106 103 113 109 113 114
± ± ± ± ± ± ± ± ± ±
5.3 2.9 4.2 2.1** 4.8* 2** 5.6 1.7** 5.6 1.7**
60 min 105 93 95 93 96 93 100 97 107 104
± ± ± ± ± ± ± ± ± ±
2.3** 2.5 4* 3.4 3.2* 3.7 2.1* 3.5 2.3* 3.5**
120 min 97 83 87 84 86 82 91 83 93 93
± ± ± ± ± ± ± ± ± ±
6.3 4.1* 5.3 6.1 2.5 2.1** 3.1 3.9 3.1* 3.9**
Each value represents mean ± S.E, n = 5; CCM, methanolic extract; CCES, ethylacetate soluble fraction of methanolic extract; CCAS, acetone soluble fraction of methanol extract; CCAIS, acetone insoluble fraction of methanolic extract. * Represents statistical significance vs. control (p < 0.05). ** Represents statistical significance vs. control (p < 0.001).
expressed as mg of rutin equivalents (RE)/g and as mg of naringin equivalents (NE)/g of extract respectively.
and were kept under regular observation for symptoms of mortality and behavioral changes for the period of 48 h.
2.5. Glucose tolerance test
2.9. Statistical analysis
The oral glucose tolerance test (Jain et al., 2010) was performed in overnight fasted (18 h) normal rats. Rats were divided into ten groups, each consisting of five rats. Group I was administered with 0.9% w/v saline; group II received metformin (50 mg/kg i.p.); Group III–X received CCM extract and its fractions in dose range of 200 and 400 mg/kg per oral. Glucose (3 g/kg) was fed 30 min after the administration of test samples. Blood was withdrawn from tail-tip at 0, 30, 60 and 120 min of glucose administration and glucose levels were estimated using glucose oxidase–peroxidase reactive strips and a glucometer (Accuchek, Roche Diagnostics, USA).
The results were expressed as mean ± SD. Comparison between the groups were made by analysis of variance (ANOVA), followed by Dunnett’s test as per suitability. p < 0.05 was considered significant.
2.6. Induction of experimental diabetes Diabetes was induced in overnight fasted rats by the intraperitoneal (i.p.) injection of alloxan monohydrate 130 mg/kg dissolved in 0.1 M cold citrate buffer (pH 4.5). The animals were allowed to drink 5% glucose solution overnight to overcome the drug induced hypoglycemia. Seventy-two hours after the injection, blood was withdrawn from overnight fasted animals and blood glucose level was assessed by glucometer. The rats with a blood glucose level above 200 mg/dl were selected for the experiment as diabetic rats (Cam et al., 2003). Control animals were injected with normal saline alone. 2.7. Experimental design The diabetic rats were fasted overnight and divided randomly into ten groups (I–X) of five rats (n = 5) each as follows, namely: Group I-Received vehicle (distilled water 10 ml/kg; p.o.); Group II-Received metformin (50 mg/kg; i.p.); Group III–X received CCM extract and its fractions in dose range of 200 and 400 mg/kg per oral. After this administration, antidiabetic activity was assessed by withdrawing blood samples at 0, 1, 3, 5 and 24 h respectively and reported the results as mg/dl (Kamalakkannan and Ponnaian, 2006). 2.8. Acute toxicity studies (Dixon, 1965; OECD, 2000) Rats were divided into test and control groups (n = 5). The test group was given an increasing oral dose (1, 3 and 5 g/kg) of CCM and its fractions. The rats were allowed food and water ad libitum
3. Results and discussion 3.1. Phytochemical screening The phytochemical screening of CCM extract revealed the presence of phenolic compounds, tannins, flavonoids, carbohydrates, alkaloids, steroids and proteins. CCES fraction has shown the presence of tannins, steroids and flavonoids. CCAS fraction showed the presence of tannins, flavonoids and proteins. While, CCAIS fraction showed the presence of carbohydrates, tannins, flavonoids and alkaloids. 3.2. Determination of total polyphenol, flavonoid and flavanones The total polyphenol content (mg/g) determined by Folin–Ciocalteu colorimetric method was found to be 15.8 ± 1.2, 18.55 ± 0.34, 25.33 ± 0.17 and 4.25 ± 0.023 mg (GAE mg/g of extract) for CCM, CCES, CCAS and CCAIS respectively. Polyphenol content was determined from linear regression equation of Gallic acid and expressed as GAE of extract (y = 0.009x + 0.099, r2 = 0.996). The flavonoid content determined by aluminum chloride method was found to be 2.92 ± 0.03, 1.534 ± 0.30, 3.46 ± 0.57, and 1.36 RE mg/g of extracts for CCM, CCES, CCAS and CCAIS respectively. Flavonoid content was determined from linear regression equation of rutin (y = 0.014x + 0.043, r2 = 0.993). The flavanone content determined by 2,4dinitrophenylhydrazine method was found to be 0.346 ± 0.001, 0.013 ± 0.002, 0.4457 ± 0.005 and 0.256 ± 0.0034 NE mg/g of extract for CCM, CCES, CCAS and CCAIS respectively. Flavanone content was determined from linear regression equation of naringin (y = 0.156x + 0.564, r2 = 0.976). As suggested by Chang et al., the flavones, flavonols and isoflavones formed complexes only with aluminum chloride, while flavanones strongly reacted only with 2,4-dinitrophenylhydrazine, so the contents determined by the two methods were added up to obtain the total flavonoid content (Chang et al., 2002). The flavonoid and flavanone content represented only 18.35% (w/w) and 2.19% (w/w) of the TP in CCM extract respectively and
P.R. Itankar et al. / Journal of Ethnopharmacology 135 (2011) 430–433
similar pattern was observed in all its fraction, suggesting that the extracts are very complex, and contain many other polyphenols such as flavanones, isoflavones, phenolic acids and tannins, and the degree of polymerization of the polyphenols present in the samples is high. Degree of polymerization can be estimated by the ratio between the TP and TFA contents (Jesus et al., 2008). The highest polymerization is observed in CCES fraction and it varies from 5.41, 12.093, 7.32 and 3.17 for CCM, CCES, CCAS and CCAIS respectively. 3.3. Antihyperglycemic effect Acute toxicity studies revealed the non-toxic nature of CCM extract and its fractions. There were no lethality or toxic reactions found at any doses selected. The CCM extract, CCES, CCAS and CCAIS fractions showed a significant reduction in blood glucose levels from 30 min onwards in oral glucose tolerance test (Table 1). The most significant antidiabetic activity was observed with metformin at dose level of 50 mg/kg i.p. While comparing with the diabetic control, amongst the extract and its fractions, CCM extract and CCES fraction have significantly lowered the elevated blood glucose levels. The most prominent antidiabetic activity was observed by CCES fraction (64.5%) at dose level of 400 mg/kg, per oral after 24 h. While the CCAS fraction marginally lowered the blood glucose level in both the selected doses (Fig. 1) and CCAIS fraction was found to be inactive at the dose level of 400 mg/kg of body weight (data not shown). Results obtained from experiment revealed that extract and its fractions have shown antidiabetic activity in decreasing order of CCES > CCM > CCAS > CCAIS. In Conclusion, the increased antidiabetic potential of CCES fraction over CCM extract is due to its partial purification achieved by fractionation which resulted in increase in degree of polymerization and segregation of secondary metabolites such as steroids and complex polyphenols present in the CCM extract. It is also evident from previous studies that with increase in degree of polymerization of extract, cytotoxic, anti-inflammatory and immunomodulatory activity increase (Young et al., 2000; Montserrat, 2007; Mursu, 2007). Suggesting that the degree of polymerization along with other secondary metabolites such as steroids and polyphenolics present in CCES fraction may play important role in its antidiabetic potential. Further study is needed to ascertain the exact mechanism of antidiabetic activity and effect of degree of polymerization on antidiabetic potential. References Anonymous, 1985. The Wealth of India, A Dictionary of Indian Raw Materials and Industrial Products. National Institute of Science and Communication (C.I.S.R.), New Delhi. Balakrishnan, N., Bhaskar, V.H., 2009. Karaunda (Carissa carandas Linn.) – as a phytomedicine: a review. The Pharma Review 9, 95–100.
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Antidiabetic potential of unripe Carissa carandas Linn. fruit extract Prakash R. Itankar ∗ , Sarika J. Lokhande 1 , Prashant R. Verma 2 , Sumit K. Arora 3 , Rajesh A. Sahu 4 , Arun T. Patil 5 Department of Pharmaceutical Sciences, Pharmacognosy and Phytochemistry Division, Rashtrasant Tukadoji Maharaj Nagpur University, Amravati Road, Nagpur 440 033, Maharashtra, India
a r t i c l e
i n f o
Article history: Received 24 October 2010 Received in revised form 10 March 2011 Accepted 14 March 2011 Available online 23 March 2011 Keywords: Antidiabetic Carissa carandas Flavonoid Flavanone Polymerization
a b s t r a c t Ethnopharmacological relevance: Carissa carandas commonly known as Karanda have a long history of use in traditional system of medicine. It is used by tribal healers of Western Ghat region of Karnataka as hepatoprotective and antihyperglycemic. However, no scientific data is available to validate the folklore claim. The present study has been designed to evaluate its unripe fruit for the antidiabetic activity. Aim: In the present study, methanol extract of unripe fruits and its fractions were studied for its antidiabetic potential. Materials and methods: The methanol extract and its fractions were screened for antidiabetic activity in alloxan induced diabetic rats. The polyphenolic, flavonoid and flavanone contents of methanolic extract and its fractions were also determined and correlated with its antidiabetic activity. Results: The experimental data indicated that the methanol extract and its ethyl acetate soluble fraction has significantly lowered the elevated blood glucose levels by 48% (p < 0.001) and 64.5% (p < 0.001) respectively at dose level of 400 mg/kg per oral after 24 h as compared to diabetic control. In order to assess the role of polyphenolic components in the relevant activity, polyphenolic and flavonoid contents were determined. The polyphenolic and flavonoid content of methanol extract and its ethyl acetate soluble fraction were found to be 15.8 ± 1.2 mg and 18.55 ± 0.34 mg (gallic acid equivalent/g extract) and flavonoid content 2.92 ± 0.03 mg and 1.534 ± 0.30 mg (rutin equivalent/g extract) respectively. Conclusion: The increased antidiabetic potential of ethyl acetate fraction over methanol extract is due to its partial purification achieved by fractionation which resulted in increase in degree of polymerization and segregation of secondary metabolites. © 2011 Published by Elsevier Ireland Ltd.
1. Introduction Diabetes mellitus (DM) is a major endocrine disorder, affecting approximately 5% of the world’s population. Diabetes is characterized by abnormalities in carbohydrate, lipid and lipoprotein metabolisms, which not only lead to hyperglycaemia but also cause many complications such as hyperlipidemia, hyperinsulinemia, hypertension and atherosclerosis (Luo et al., 2004; Sepici et al., 2004).
∗ Corresponding author. Tel.: +91 712 2550324; fax: +91 712 2500355; mobile: +91 9423677401. E-mail addresses: pri [email protected] (P.R. Itankar), [email protected] (S.J. Lokhande), [email protected] (P.R. Verma), sum [email protected] (S.K. Arora), [email protected] (R.A. Sahu), [email protected] (A.T. Patil). 1 Mobile: +91 8928002537. 2 Mobile: +91 9423100313. 3 Mobile: +91 9960703930. 4 Mobile: +91 9373102633. 5 Mobile: +91 9422808227. 0378-8741/$ – see front matter © 2011 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2011.03.036
Carissa carandas commonly known as Karanda belongs to family Apocynaceae. In recent years its botanical name was changed to Carissa congesta Wight (syn. Carissa carandas Auct. Formerly widely known as Carissa carandas L.) (Anonymous, 1985). Carissa carandas is large dichotomously branched evergreen shrub with short stem and strong thorn in pairs. This species is a rank-growing, straggly, woody, climbing shrub, usually growing to 10 or 15 ft (3–5 m) high, sometimes ascending to the tops of tall trees. The plant is native and common throughout India (Kirtikar and Basu, 2003). Traditionally, whole plant and its parts were used in the treatment of various ailments. The roots were employed as a bitter stomachic, vermifuge and as an ingredient in the remedy for itches. The roots were reported to contain salicylic acid and cardiac glycosides. It also contains carissone; d-glycoside of -sitosterol; glucosides of odoroside H; carindone; a terpenoid lupeol; ursolic acid and its methyl ester; also carinol, a phenolic lignan. Fruits contains good amount of vitamin C. The fruits, leaves and bark are rich in tannins (Morton, 1987). In Ayurveda, the unripe fruits were used as an anthelmintic, astringent, appetizer, antipyretic, antidiabetic, aphrodisiac, in biliary disorders, stomach disorders, rheumatism and diseases of the
P.R. Itankar et al. / Journal of Ethnopharmacology 135 (2011) 430–433
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Fig. 1. Effect of Carissa carandas fruit extract and fractions on diabetic animals. Each value represents mean ± S.E., n = 5; CCM, methanolic extract; CCES, ethylacetate soluble fraction of methanolic extract; CCAS, acetone soluble fraction of methanol extract; CCAIS, acetone insoluble fraction of methanolic extract. *Represents statistical significance vs. control (p < 0.05). **Represents statistical significance vs. control (p < 0.001).
brains (Morton, 1987; Iyer and Dubhash, 2006). It is useful in treatment of diarrhea, anorexia and intermittent fevers. Fruits have also been studied for its analgesic, anti-inflammatory and lipase 1 activities (Balakrishnan and Bhaskar, 2009). It is used by tribal healers of Western Ghat region of Karnataka as hepatoprotective and antihyperglycemic. However, no scientific data is available to validate the folklore claim (Kirtikar and Basu, 2003; Christophe, 2006). Keeping the above information in view, the present study has been designed to evaluate its antidiabetic activity. 2. Materials and methods 2.1. Animals Swiss albino rats of Sprague–Dawley strain (200–250 g) of either sex obtained from animal house of our institute were used. The animals were fed a standard pellet diet and water ad libitum. They were maintained in a controlled environment and temperature (22 ± 5 ◦ C with 12-h of light/dark cycle). All experimental protocols were approved by the Institutional Animal Ethical Committee (12/2009/CPCSEA). 2.2. Chemicals and standard drugs Aluminum trichloride hexahydrate, glacial acetic acid, anhydrous sodium carbonate, Folin–Ciocalteu phenol reagent, naringin, gallic acid, rutin (hydrate, min 95%), sodium acetate and 2,4dinitrophenylhydrazine were of analytical grade obtained from S. D. Fine Chemicals Pvt. Ltd. (Vadodra, India). 2.3. Plant material, preparation of extract and its fractionation The fruits were collected locally, authenticated by the Department of Botany, R. T. M. Nagpur University Campus, Nagpur. A voucher specimen has been deposited in the Herbarium of Department of Botany, with collection number RA 9496. The fruits were dried under shade and pulverized to a coarse powder. The powdered crude material was defatted with petroleum ether and then extracted with methanol. The methanol extract (CCM) was concentrated in a rotary vacuum evaporator to yield a dark brown mass (yield 28.07%, w/w). For fractionation, methanol extract was triturated with silica (1:3), loaded to Soxhlet assembly and extracted by ethyl acetate to yield ethyl acetate soluble fraction (CCES; yield: 21.65%, w/w) (Fuentes and Alarcón, 2006). The ethyl acetate insoluble portion was further extracted with acetone to yield acetone soluble fraction (CCAS; yield 10.56%,
w/w) and acetone insoluble fraction (CCAIS; yield: 67.19%, w/w). The methanol extract (CCM) and these three broad fractions i.e. CCES, CCAS and CCAIS were subjected to phytochemical and pharmacological screening. 2.4. Phytochemical screening (Stahl, 1969; Harbone, 1976; Wagner et al., 1984) The CCM extract and its broad fractions were screened for the presence of tannins, saponins, unsaturated sterols, triterpenes, alkaloids, flavonoids, lactones/esters, protein/amino acids and carbohydrates and/or glycosides with thin layer chromatography (TLC). Thin layer plates precoated with silica gel G (Merck, 0.25 mm thickness) were used. Development was carried out with different solvent systems such as ethyl acetate:methanol:water (100:13.5:10, v/v/v), ethyl acetate:formic acid:acetic acid:water (100:11:11:26, v/v/v/v), chloroform:methanol:water (70:30:4, v/v/v), toluene:ethyl acetate:diethylamine (70:20:10, v/v/v) and ethyl acetate:methanol:water:acetic acid (65:15:15:10, v/v/v/v). After development of chromatogram in the solvents the plates were dried and sprayed with Dragendorff’s, AlCl3 , hydroxylamine-ferric chloride, ninhydrin and antimony trichloride for the detection of alkaloids, flavonoids, lactones/esters, protein/amino acids, unsaturated sterols and triterpenes respectively. While detection of anthraquinones, saponins, tannins, carbohydrate and/or glycosides is carried out using KOH, anisaldehyde-sulphuric acid reagent, ferric chloride and naphthoresorcinol reagent respectively and visualization is carried out under visible and UV light (: 366 nm). CCM extract and its fractions were also quantified for presence of important secondary metabolites such as total polyphenol, flavonoid and flavonone compounds using following spectroscopic methods. 2.4.1. Determination of total polyphenol compounds (TP), flavonoids (TFA) and total flavanones (TFO) Total polyphenol content was measured using Folin–Ciocalteu colorimetric method (Singleton and Rossi, 1965; Singlenton et al., 1999). Gallic acid was used as a reference for constructing the standard curve (10–100 mg/ml). The results were expressed as mg of gallic acid equivalents (GAE)/g of extract. Flavonoid content was determined by the aluminum chloride method (Jin-Yuarn and Ching-Yin, 2007; Stanojevic´ et al., 2009). While the modified 2,4-dinitrophenylhydrazine method was used for determination of flavanones (Nagy and Grancai, 1996). Rutin and naringin were used as a reference standard and results were
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Table 1 Effect of Carissa carandas fruit extract and fractions on oral glucose tolerance test. Group
Extract/fractions
Blood glucose concentration (mg/dl)
I II III IV V VI VII VIII IX X
Control Metformin (50 mg/kg i.p.) CCM extract (200 mg/kg p.o.) CCM extract (400 mg/kg p.o.) CCES fraction (200 mg/kg p.o.) CCES fraction (400 mg/kg p.o.) CCAS fraction (200 mg/kg p.o.) CCAS fraction (400 mg/kg p.o.) CCAIS fraction (200 mg/kg p.o.) CCAIS fraction (400 mg/kg p.o.)
89 83 85 85 83 82 87 84 87 86
0 min ± ± ± ± ± ± ± ± ± ±
1.7* 1** 3.1* 3* 1.9 2.1** 3.1 2.2** 3.1** 2.2*
30 min 113 99 102 101 106 103 113 109 113 114
± ± ± ± ± ± ± ± ± ±
5.3 2.9 4.2 2.1** 4.8* 2** 5.6 1.7** 5.6 1.7**
60 min 105 93 95 93 96 93 100 97 107 104
± ± ± ± ± ± ± ± ± ±
2.3** 2.5 4* 3.4 3.2* 3.7 2.1* 3.5 2.3* 3.5**
120 min 97 83 87 84 86 82 91 83 93 93
± ± ± ± ± ± ± ± ± ±
6.3 4.1* 5.3 6.1 2.5 2.1** 3.1 3.9 3.1* 3.9**
Each value represents mean ± S.E, n = 5; CCM, methanolic extract; CCES, ethylacetate soluble fraction of methanolic extract; CCAS, acetone soluble fraction of methanol extract; CCAIS, acetone insoluble fraction of methanolic extract. * Represents statistical significance vs. control (p < 0.05). ** Represents statistical significance vs. control (p < 0.001).
expressed as mg of rutin equivalents (RE)/g and as mg of naringin equivalents (NE)/g of extract respectively.
and were kept under regular observation for symptoms of mortality and behavioral changes for the period of 48 h.
2.5. Glucose tolerance test
2.9. Statistical analysis
The oral glucose tolerance test (Jain et al., 2010) was performed in overnight fasted (18 h) normal rats. Rats were divided into ten groups, each consisting of five rats. Group I was administered with 0.9% w/v saline; group II received metformin (50 mg/kg i.p.); Group III–X received CCM extract and its fractions in dose range of 200 and 400 mg/kg per oral. Glucose (3 g/kg) was fed 30 min after the administration of test samples. Blood was withdrawn from tail-tip at 0, 30, 60 and 120 min of glucose administration and glucose levels were estimated using glucose oxidase–peroxidase reactive strips and a glucometer (Accuchek, Roche Diagnostics, USA).
The results were expressed as mean ± SD. Comparison between the groups were made by analysis of variance (ANOVA), followed by Dunnett’s test as per suitability. p < 0.05 was considered significant.
2.6. Induction of experimental diabetes Diabetes was induced in overnight fasted rats by the intraperitoneal (i.p.) injection of alloxan monohydrate 130 mg/kg dissolved in 0.1 M cold citrate buffer (pH 4.5). The animals were allowed to drink 5% glucose solution overnight to overcome the drug induced hypoglycemia. Seventy-two hours after the injection, blood was withdrawn from overnight fasted animals and blood glucose level was assessed by glucometer. The rats with a blood glucose level above 200 mg/dl were selected for the experiment as diabetic rats (Cam et al., 2003). Control animals were injected with normal saline alone. 2.7. Experimental design The diabetic rats were fasted overnight and divided randomly into ten groups (I–X) of five rats (n = 5) each as follows, namely: Group I-Received vehicle (distilled water 10 ml/kg; p.o.); Group II-Received metformin (50 mg/kg; i.p.); Group III–X received CCM extract and its fractions in dose range of 200 and 400 mg/kg per oral. After this administration, antidiabetic activity was assessed by withdrawing blood samples at 0, 1, 3, 5 and 24 h respectively and reported the results as mg/dl (Kamalakkannan and Ponnaian, 2006). 2.8. Acute toxicity studies (Dixon, 1965; OECD, 2000) Rats were divided into test and control groups (n = 5). The test group was given an increasing oral dose (1, 3 and 5 g/kg) of CCM and its fractions. The rats were allowed food and water ad libitum
3. Results and discussion 3.1. Phytochemical screening The phytochemical screening of CCM extract revealed the presence of phenolic compounds, tannins, flavonoids, carbohydrates, alkaloids, steroids and proteins. CCES fraction has shown the presence of tannins, steroids and flavonoids. CCAS fraction showed the presence of tannins, flavonoids and proteins. While, CCAIS fraction showed the presence of carbohydrates, tannins, flavonoids and alkaloids. 3.2. Determination of total polyphenol, flavonoid and flavanones The total polyphenol content (mg/g) determined by Folin–Ciocalteu colorimetric method was found to be 15.8 ± 1.2, 18.55 ± 0.34, 25.33 ± 0.17 and 4.25 ± 0.023 mg (GAE mg/g of extract) for CCM, CCES, CCAS and CCAIS respectively. Polyphenol content was determined from linear regression equation of Gallic acid and expressed as GAE of extract (y = 0.009x + 0.099, r2 = 0.996). The flavonoid content determined by aluminum chloride method was found to be 2.92 ± 0.03, 1.534 ± 0.30, 3.46 ± 0.57, and 1.36 RE mg/g of extracts for CCM, CCES, CCAS and CCAIS respectively. Flavonoid content was determined from linear regression equation of rutin (y = 0.014x + 0.043, r2 = 0.993). The flavanone content determined by 2,4dinitrophenylhydrazine method was found to be 0.346 ± 0.001, 0.013 ± 0.002, 0.4457 ± 0.005 and 0.256 ± 0.0034 NE mg/g of extract for CCM, CCES, CCAS and CCAIS respectively. Flavanone content was determined from linear regression equation of naringin (y = 0.156x + 0.564, r2 = 0.976). As suggested by Chang et al., the flavones, flavonols and isoflavones formed complexes only with aluminum chloride, while flavanones strongly reacted only with 2,4-dinitrophenylhydrazine, so the contents determined by the two methods were added up to obtain the total flavonoid content (Chang et al., 2002). The flavonoid and flavanone content represented only 18.35% (w/w) and 2.19% (w/w) of the TP in CCM extract respectively and
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similar pattern was observed in all its fraction, suggesting that the extracts are very complex, and contain many other polyphenols such as flavanones, isoflavones, phenolic acids and tannins, and the degree of polymerization of the polyphenols present in the samples is high. Degree of polymerization can be estimated by the ratio between the TP and TFA contents (Jesus et al., 2008). The highest polymerization is observed in CCES fraction and it varies from 5.41, 12.093, 7.32 and 3.17 for CCM, CCES, CCAS and CCAIS respectively. 3.3. Antihyperglycemic effect Acute toxicity studies revealed the non-toxic nature of CCM extract and its fractions. There were no lethality or toxic reactions found at any doses selected. The CCM extract, CCES, CCAS and CCAIS fractions showed a significant reduction in blood glucose levels from 30 min onwards in oral glucose tolerance test (Table 1). The most significant antidiabetic activity was observed with metformin at dose level of 50 mg/kg i.p. While comparing with the diabetic control, amongst the extract and its fractions, CCM extract and CCES fraction have significantly lowered the elevated blood glucose levels. The most prominent antidiabetic activity was observed by CCES fraction (64.5%) at dose level of 400 mg/kg, per oral after 24 h. While the CCAS fraction marginally lowered the blood glucose level in both the selected doses (Fig. 1) and CCAIS fraction was found to be inactive at the dose level of 400 mg/kg of body weight (data not shown). Results obtained from experiment revealed that extract and its fractions have shown antidiabetic activity in decreasing order of CCES > CCM > CCAS > CCAIS. In Conclusion, the increased antidiabetic potential of CCES fraction over CCM extract is due to its partial purification achieved by fractionation which resulted in increase in degree of polymerization and segregation of secondary metabolites such as steroids and complex polyphenols present in the CCM extract. It is also evident from previous studies that with increase in degree of polymerization of extract, cytotoxic, anti-inflammatory and immunomodulatory activity increase (Young et al., 2000; Montserrat, 2007; Mursu, 2007). Suggesting that the degree of polymerization along with other secondary metabolites such as steroids and polyphenolics present in CCES fraction may play important role in its antidiabetic potential. Further study is needed to ascertain the exact mechanism of antidiabetic activity and effect of degree of polymerization on antidiabetic potential. References Anonymous, 1985. The Wealth of India, A Dictionary of Indian Raw Materials and Industrial Products. National Institute of Science and Communication (C.I.S.R.), New Delhi. Balakrishnan, N., Bhaskar, V.H., 2009. Karaunda (Carissa carandas Linn.) – as a phytomedicine: a review. The Pharma Review 9, 95–100.
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