Fitoterapia 83 (2012) 142–146
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Anti-diabetic potential of ursolic acid stearoyl glucoside: A new triterpenic gycosidic ester from Lantana camara Imran Kazmi a, Mahfoozur Rahman b, Muhammad Afzal a, Gaurav Gupta a, Shakir Saleem c, Obaid Afzal c, Md.Adil Shaharyar c, Ujjwal Nautiyal a, Sayeed Ahmed c, Firoz Anwar a,⁎ a b c
Siddhartha Institute of Pharmacy, Uttarakhand, India Dreamz College of Pharmacy, Himachal Pradesh, India Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
a r t i c l e
i n f o
Article history: Received 27 June 2011 Accepted in revised form 10 October 2011 Available online 24 October 2011 Keywords: Lantana camara Stearoyl glucoside of ursolic acid Anti-diabetic
a b s t r a c t A new stearoyl glucoside of ursolic acid, urs-12-en-3β-ol-28-oic acid 3β-D-glucopyranosyl-4′octadecanoate and other compounds were isolated from the leaves of Lantana camara L. The structure of this new glycoside was elucidated and established by standard spectroscopic methods. In streptozotocin-induced diabetic rats it showed significant reduction in blood glucose level. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
1. Introduction Lantana camara L. is regarded as a notorious weed; however it is popular as an ornamental garden plant and is used in folk medicine worldwide [1]. It is known as Red or Yellow or Wild sage and has been used in many parts of the world to treat a wide variety of disorders [2,3]. In spite of its medicinal importance, it is an invasive plant species that has been found in diverse geophysical environments, causing a threat to the native flora. Various eradication programmes have been attempted such as burning, chemical sprays, biocontrol agents and physical plugging mechanism to remove such invasive species in India, Australia and many other countries [4–6]. Tea or decoction prepared from leaves and flowers is used as a remedy and treatment for cough, fever, influenza, stomach ache, tetanus, rheumatism, malaria and ataxy of abdominal viscera [7,8]. Pounded leaves are applied to cuts, ulcers and swellings for their healing properties [9]. Extract of this ⁎ Corresponding author. Tel.: + 91 9412937329; fax: + 91 135 2607784. E-mail address: fi
[email protected] (F. Anwar).
plant is used in folk medicine for the treatment of cancers, chicken pox, measles, asthma, ulcers, swellings, eczema, tumors, high blood pressure, bilious fevers, catarrhal infections, tetanus, rheumatism, malaria and ataxy of abdominal viscera [1,10]. The plant has displayed anticonvulsant [11], termicidal [12], wound healing [13,14], anticancer [15,16], antiulcer [17], antioxidant [16], anti-diabetic [18,19], analgesic and anti-inflammatory [20], anti-motility [21], anti-feedant, larval mortality/repellency [22–24], antifungal and antibacterial [25–27] activities. The leaves are found to contain various chemical constituents such as monoterpenes; sesquiterpenes including germacrene-D, γ-elemene, β-caryophyllene, β-elemene, α-copaene and αcadinene [28], zingiberene [29], b-curcumene, (E)-nuciferal, (Z)-nuciferol, (−)ar-curcumen-15-al, g-curcumene, arcurcumene, (−)-γ-epi-b-bisabolol and (−)-γ-curcumen-15-al [30, 31]; triterpenes including lantadenes [32–35], lantic acid [36], camarolic acid, lantrigloylic acid, camaric acid, lantanolic acid, lantanilic acid, pomolic acid, camarinic acid, lantoic acid, camarin, lantacin, camarinin, ursolic acid [37] and 28noroleanane [38]; glycosides including theveside, 3-methoxy-, 3,7-dimethoxyand 3,7,49-trimethoxyquercetin [39],
0367-326X/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.10.004
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camaraside [40], verbacoside [41], martynoside [1], derhamnosylverbascoside, isonuomioside A and an iridoid glycoside [42,43]. The leaves are a rich source of fatty acids [44]; proteins [45] and carbohydrates [46]. 2. Materials and methods 2.1. Plant material L. camara Linn. leaves were collected from Hamdard University and identified by Dr. S. B. Singh, Scientist, NISCAIR, New Delhi. A voucher specimen (NISCAIR/RHMD/consult/20-09-10/1322/125) was deposited in the herbarium of NISCAIR, India. 2.2. Extraction and isolation procedure Dried powder of L. camara leaves (1.7 kg) was extracted with methanol (4 L) at 50 °C for 1 day. Extract was concentrated to dryness under reduced pressure to obtain slurry (270 g). The slurry was dissolved in minimum amount of methanol and was adsorbed on silica gel (60–120 mesh). The slurry was subjected to a silica gel column using a petroleum ether/CHCl3/MeOH gradient system (1:0:0, 3:1:0, 1:1:0, 1:3:0, 0:1:0, 0:99:1, 0:49:1, 0:97:3, 0:19:1, 0:97:7, 0:91:9, 0:89:11, 0:87:13 and 0:17:3; 2.0 L for each gradient system). A new compound 1 (270 mg) from CHCl3/MeOH (49:1) along with two other known compounds, compound 2 from CHCl3/ MeOH (97:3) and compound 3 from CHCl3/MeOH (93:7) were obtained as colourless crystals. 2.2.1. urs-12-en-3β-ol-28-oic acid 3β-D-glucopyranosyl-4′octadecanoate (1) Colourless crystal, mp 281–282 C; Rf: 0.28 (CHCl3/MeOH, 99:1); UV λmax (MeOH): 220 nm (log ε 4.8); IR γmax (KBr): 3424, 3392, 3150, 2930, 2855, 1721, 1703, 1635, 1447, 1379, 1223, 1183, 1096, 1035, 994, 767 cm− 1; 1H NMR (CDCl3): δ 5.35 (1H, d, J = 6.0 Hz, H-12), 5.01 (1H, d, J = 7.2 Hz, H-1′), 4.67 (1H, m, H-5 ), 4.32 (1H, dd, J = 7.2, 5.1 Hz, H-2′), 4.26 (1H, m, H-3′), 4.15 (1H, m, H-4′), 3.62 (1H, dd, J = 5.3, 9.6 Hz, H-3α), 3.39 (1H, d, J = 8.7 Hz, H2-6′a), 3.25 (1H, d, J = 9.3 Hz, H2-6b′), 2.40 (1H, d, J = 7.8 Hz, H2-2a), 2.35 (1H, d , J = 8.4 Hz, H2-2b), 2.03 (2H, m, CH2), 1.61(4H, m, 2 × CH2), 1.58 (4H, m, 2 × CH2), 1.28 (6H, brs, 3 × CH2), 1.25 (14H, brs, 7 × CH2), 1.08 (3H, brs, Me-23), 1.03 (3H, brs, Me-27), 0.97 (3H, brs, Me-25), 0.95 (3H, brs, Me-24), 0.92 (3H, d, J = 6.1 Hz, Me-29), 0.89 (3H, brs, Me-26), 0.85 (3H, t,
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J = 6.2 Hz, Me-18 ), 0.76 (3H, d, J = 6.3 Hz, Me-30); MS m/z: 884 [M]+ (C54H92O9). 2.3. Anti-diabetic activity 2.3.1. Animals Wistar albino rats (150–200 g) were obtained from Central Animal Facility, Hamdard University and kept at 25± 1 °C, 55 ± 5% humidity along with 12 h light/dark cycle. The animals were given standard pellet diet (Lipton rat feed, Ltd., Pune) and water ad libitum throughout the experimental period. The experiment was approved by the ‘Institutional Animal Ethics Committee’. Extract of compound (1) and the standard drug were administered orally. 2.3.2. Chemicals Only analytical grades chemicals and reagents were used. Streptozotocin (Spectrochem Pvt. Ltd. Mumbai, India) was obtained from Chopra chemicals (Delhi, India). 2.3.3. Drugs Standard drug: Glimepiride was prepared in Tween 80 solution; Test drug: plant extract in CMC (1%) solution and compound 1 in Tween 80 solution. 2.3.4. Induction of diabetes The animals were fasted for 16 h prior to the induction of diabetes. STZ freshly prepared in citrate buffer (pH 4.5) was administered i.p. as a single dose of 50 mg/kg body weight. The development of diabetes was confirmed by polydipsia, polyurea and by measuring blood glucose concentrations 72 h after injection of STZ. The rats with blood glucose level of 250 mg/dl or higher were considered to be diabetic and were selected for the experiment. The animals were randomly assigned to various groups. Group I was the normal untreated control rats and received distilled water daily for 21 days. Group 2, the diabetic control rats, received distilled water during the experiment. Animals of Group III received the reference standard drug Glimepiride (0.1 mg/kg), Groups IV to V received the aqueous extracts of L. camara (250 mg/ kg, and 500 mg/kg) and Group VI received Compound 1 (0.3 mg/kg). 2.3.5. Estimation of blood glucose The non fasting blood glucose level was determined just before administering the drugs on 1st, 7th, 14th and 21st day. The blood glucose level was estimated with One Touch
Table 1 Antidiabetic activity of compound 1 on Wister albino rat. Groups
Treatment
Dose
I II III IV V VI
Normal Diabetic control (streptozotocin) Standard (Glimepiride) Aq. extract of L. camara Aq. extract of L. camara Compound I
Normal saline 50 mg/kg 0.1 mg/kg 250 mg/kg 500 mg/kg 0.3 mg/kg
Blood glucose level in mg/dl 1st day
8st day
14st day
21st day
113.73 ± 4.69 370.33 ± 5.05 374.67 ± 5.06 366.67 ± 5.80 361.83 ± 14.72 366.45 ± 5.89
114.67 ± 1.02 374.50 ± 1.76## 161.33 ± 2.21⁎⁎ 183.83 ± 4.29⁎⁎ 180.50 ± 3.07⁎⁎ 183.56 ± 3.61⁎⁎
116.50 ± 1.56 368.33 ± 3.22## 124.67 ± 1.80⁎⁎ 165.50 ± 4.26⁎⁎ 157.83 ± 5.28⁎⁎ 143.43 ± 2.79⁎⁎
119.50 ± 1.15 375.33 ± 4.46## 105.50 ± 3.12⁎⁎ 136.83 ± 1.99⁎⁎ 124.67 ± 2.40⁎⁎ 118.67 ± 2.40⁎⁎
Values are given as mean ± S.E.M. of six rats in each group. ## (P b 0.01) compared with the corresponding value for normal control animals (group I); ** (P b 0.01) compared with the corresponding value for diabetic control animals (group II).
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Basic Glucometer (Accu Chek Active, Roche, Germany), see Table 1. 2.3.6. Statistical analysis All the data were expressed as mean ± S.E.M., and analysis of variance (ANOVA) was used for the statistical analysis. The values were considered to be significant when the P value was b0.01.
12-en-3β-ol-28-oic acid 3β-D-glucopyranosyl-4′-octadecanoate (Fig. 1). Compound 1 was evaluated for anti-diabetic activity [52– 54]. Many plant products can exert specific medicinal actions. The compounds in these plants have been explored for their therapeutic potential to prevent and cure common and lethal
3. Result and discussion The known compounds urs-12-en-3β-ol-28-oic acid (2) [47] and oleane-12-en-3β-ol-28-oic acid 3β-D-glucopyranoside (3) [48], were identified by comparison of their spectroscopic data from the reported literature. Compound 1, named stearoyl glucoside of ursolic acid was obtained as colourless crystals using chloroform-methanol (49:1) elutant. Liebermann–Burchard, Salkowski and Antimony trichloride tests [49] were positive for triterpenic glycoside. Carboxylic group in molecule was confirmed by sodium bicarbonate effervescence test. The presence of acyl group in the compound was confirmed by alkaline hydrolysis which yielded triterpenic glycoside and stearic acid [50]. The presence of acyl group was further confirmed by reaction of compound 1 with potassium hydroxide, refluxed for 20 min and distilled with ethyl alcohol. The presence of acyl group was confirmed by Iodoform test with distillate [51]. IR spectra showed the presence of hydroxyl group (3424, 3392 cm − 1), carbonyl group (3150, 1690 cm − 1), ester group (1721 cm − 1) and unsaturation (1635 cm − 1). The molecular weight of compound was established as 884 which was found consistent with the molecular formula of pentacyclic triterpene glycosidic ester; C54H92O9. Nine degrees of unsaturation, five of them adjusted in pentacyclic carbon framework of the oleanene-type space triterpene and one of each in the vinylic linkage, ester group, sugar unit and carboxylic group were found. The 1H NMR spectra displayed two one–proton doublets at δ 5.35 (J = 6.0 Hz) and 5.01 (J = 7.2 Hz) assigned to vinylic H-12 and anomeric H-1′ protons, respectively. Three one-proton multiplets at δ 4.67, 4.26 and 4.15 and a one-proton double doublet at δ 4.32 (J = 7.2, 5.1 Hz) were ascribed correspondingly to the carbinol proton of sugar unit H-5′, H-3′, H-4′, and H-2′. The shifting of H-4 proton signal in the deshielded region suggested location of the ester group at C-4′. Four one-proton doublet at δ 3.29 (J = 7.8 Hz), 3.25 (J = 9.3 Hz), δ 2.40 (J = 7.8 Hz) and at 2.35 (J = 8.4 Hz) were attributed to hydroxy methylene H2-6 and the methylene H2-2 protons adjacent to the ester group. Five proton triplets at δ 1.08, 1.03, 0.94, 0.95, and 0.89 were due to tertiary C-23, C-27 C-25, C-24, and C-26 methyl protons respectively, all attached to saturated carbons. Two three-proton doublets at δ 0.92 (J = 6.1 Hz) and 0.76 (J = 6.3 Hz) and a three-proton triplet at δ 0.85 (J = 6.2 Hz) accounted for secondary C-29 and C-30 and primary C-18 methyl protons, respectively. The remaining methylene and methane proton appeared between δ 2.83 and 1.25. Acid hydrolysis of compound 1 yielded ursolic acid, D-glucose and stearic acid, comparable with co-TLC. On the basis of spectral data analysis and chemical reactions, the structure of new triterpenic gycosidic ester was established as urs-
1
2
3 Fig. 1. Structures of compounds 1–3.
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metabolic disorders like diabetes, obesity etc. The presence of new triterpenoid glycoside in L. camara prompted authors to go for anti-diabetic evaluation of this new compound. The study conducted clearly depicts the anti-diabetic potential of compound 1, reflecting its sugar lowering property in the experimental rats from 8th day to the 21st day of the experiment (Table 1). The result (p b 0.01) was comparatively promising when Glimepiride was used as a standard drug. Among the number of terpenoids which have anti-diabetic potential [55–57], very few have been tried for in-vivo studies. The establishment of anti-diabetic activity for compound 1 requires further research and its exact mechanism of action needs to be deciphered. Till date no triterpenoids formulation is commercially available. If properly explored in terms of its specific mechanism of action, this might prove as a novel triterpenoid with anti-diabetic properties. Acknowledgements The authors are humbly thankful to Mr. Durga Verma (Chairman) and Mr. R.R. Aggarwal (Director) of Siddhartha Institute of Pharmacy, Dehradun, India and NIMS University, Jaipur, Rajasthan, India for providing lab and library facilities. References [1] Ghisalberti EL. Lantana camara L. (Verbenaceae). Fitoterapia 2000;71: 467–86. [2] Sastri CST, Kavathekar KY. Plants for reclamation of wastelands. New Delhi, India: Pbl. P & I Directorate, CSIR; 1990. p. 296–8. [3] Ross IA. Medicinal plants of the world. Chemical constituents, traditional and modern medical uses. New Jersey: Humana Press; 1999. [4] Kandwal R, Jeganathan C, Tolpekin V, Kushwaha SPS. Discriminating the invasive species, ‘Lantana’ using vegetation indices. J Indian Soc Remote Sens 2009;37:275–90. [5] Rogers M, Johnson K. Stemming the spread — the lantana containment zone project. Sixteenth Australian Weeds Conference; 2008. [6] Gooden B, French K, Turner PJ, Downey PO. Impact threshold for an alien plant invader, Lantana camara L., on native plant communities. Biol Conserv 2009;142:2631–41. [7] Watt JM, Breyer-Brandwijk MG. The medicinal and poisonous plants of southern and eastern Africa. E & S Livington Ltd; 1962. p. 1947. [8] Irvine FR. Woody plants of Ghana. London: Oxford University Press; 1961. [9] Anonymous. The wealth of India, vol. 6. New Delhi: Publication and Information Directorate; 1962. p. 34. [10] Day MD, Wiley CJ, Playford J, Zalucki MP. Lantana: current management, status and future prospects. Australian Centre for International Agricultural Research: Canberra; 2003. [11] Adesina SK. Studies on some plants used as anticonvulsants in Amerindian and African traditional medicine. Fitoterapia 1982;53:147–62. [12] Verma RK, Verma SK. Phytochemical and termiticidal study of Lantana camara var. aculeata leaves. Fitoterapia 2006;77:466–8. [13] Nayak BS, Raju SS, Ramsubhag A. Investigation of wound healing activity of Lantana camara L. in Sprague Dawley rats using a burn wound model. Int J Appl Res Nat Prod 2008;1:15–9. [14] Nayak BS, Raju SS, Eversley M, Ramsubhag A. Evaluation of wound healing activity of Lantana camara L. — a preclinical study. Phytother Res 2009;23:241–5. [15] Bisi-Johnson MA, Obi CL, Hattori T, Oshima Y, Li S, Kambizi L, et al. Evaluation of the antibacterial and anticancer activities of some South African medicinal plants. BMC Complement Altern Med 2011;11:14–8. [16] Gomes de Melo J, de Sousa Araújo TA, Thijan Nobre de Almeida e Castro V, Lyra de Vasconcelos Cabral D, do Desterro Rodrigues M, Carneiro do Nascimento S, et al. Antiproliferative activity, antioxidant capacity and tannin content in plants of semi-arid northeastern Brazil. Molecules 2010;15:8534–42. [17] Sathish R, Vyawahare B, Natarajan K. Antiulcerogenic activity of Lantana camara leaves on gastric and duodenal ulcers in experimental rats. J Ethnopharmacol 2011;134:195–7.
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