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Extraction and recovery of karanjin: A value addition to karanja (Pongamia pinnata) seed oil
Industrial Crops and Products 32 (2010) 118–122
Contents lists available at ScienceDirect
Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Extraction and recovery of karanjin: A value addition to karanja (Pongamia pinnata) seed oil Vismaya a , W. Sapna Eipeson a , J.R. Manjunatha b , P. Srinivas b , T.C. Sindhu Kanya a,∗ a
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Council of Scientific & Industrial Research, Mysore 570020, India Department of Plantation Products, Spices and Flavour Technology, Central Food Technological Research Institute, Council of Scientific & Industrial Research, Mysore 570020, India b
a r t i c l e
i n f o
Article history: Received 29 August 2009 Received in revised form 21 March 2010 Accepted 30 March 2010
Keywords: Pongamia pinnata Karanja Karanjin Recovery Characterization NMR
a b s t r a c t Karanja (Pongamia pinnata) seed oil contains karanjin, a bioactive molecule with important biological attributes. The objective of this study was to develop a facile method for efficient recovery of karanjin. The seed oil was subjected to liquid–liquid extraction with methanol. The extract was further purified by chromatography on alumina followed by crystallization to afford karanjin, whose purity was ascertained by HPLC. The recovery of karanjin was ∼20% with >95% purity. The structure of the compound was elucidated by MS and NMR spectral analysis. Mass spectrum for the compound in ESI+ mode showed signals at 293 and 315 corresponding to (M+H+ ) and (M+Na+ ) adducts, respectively, confirming the molecular weight to be 292. 1 H and 13 C spectral data established the structure of the molecule to be a furanoflavonoid compound with the furan ring signals appearing at 7.78 (H␣ )/145.4 (C␣ ) and 7.20 (H )/103.9 (C ). 1 H COSY, HMBC (H → C) and DEPT spectral data showed the annelation of furan and flavonoid rings to be at C7/C8 and C9/C10 positions, respectively, and a phenyl ring substitution at 2 (1 ) position. Karanjin’s recovery in pure form and high yield affords value addition to the seed oil for pharmaceutical applications. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Many unconventional oilseeds from forest produce have been explored to overcome the shortage and high price of oils from traditional sources. They find several uses and one such seed is from Pongamia pinnata (L.) Pierre (Leguminaceae, Papilionaceae; synonym Pongamia glabra Vent.), popularly known as ‘Karanj’ or ‘Karanja’, which has received greater attention (CSIR, 1969). This medium-size tree is indigenous to the Indian subcontinent and south-east Asia. It is one of the widely grown forest trees with 0.11 million tons of seeds collected every year in India, by states like Andhra Pradesh, Karnataka and Tamil Nadu (Vinay and Sindhu Kanya, 2008). Historically, this plant has long been used in India and neighboring regions as a source of traditional medicine, animal fodder, green manure, timber and fuel. Karanja seed containing 33–36% oil is also a rich source of protein (20–28%). The oil from P. pinnata has the potential to provide an environmentally acceptable, alternative to conventional diesel engine fuels (Raheman and Phadatare, 2004). At present, karanja oil is being explored mainly for its use as bio-diesel (De and Bhattacharya, 1999; Maher et al., 2006). The de-oiled cake of P. pinnata seeds obtained after solvent
∗ Corresponding author. Tel.: +91 821 2515331; fax: +91 821 2517233. E-mail address: [email protected] (T.C. Sindhu Kanya). 0926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2010.03.011
extraction contains up to 30% protein. While it has the potential to be used as sustainable animal feed supplement, its anti-nutritional factors restrict its usage as a feed/food. Indeed, nutritional quality of karanja seed proteins was improved after reducing the antinutrients in the meal (Vinay and Sindhu Kanya, 2008). Karanja seed is used as a medicinal plant, particularly with the Ayurvedic and Sidda medicine systems of India (Muthu et al., 2006). Crude seed extract can completely inhibit the growth of herpes simplex virus type 1 and type 2 in Vero cells (Elanchezhiyan et al., 1993) and also possesses hypoglycemic, anti-oxidative, anti-ulcerogenic, anti-inflammatory and analgesic properties (Dahanukumar et al., 2000). Karanja oil contains 5–6% flavonoids (Bringi, 1987), the main constituents being karanjin, a furano-flavonoid and pongamol, a diketone. Karanjin has been isolated during the course of exploration of newer compounds from karanja seeds. Earlier works of Rao and Rao (1941) have led several workers (Aneja et al., 1963; Roy et al., 1977; Pathak et al., 1983), to identify some new components of Pongamia seed oil apart from karanjin. Many biological activities of P. pinnata seed extracts can be attributed to karanjin, the major flavonoid of the seed oil. Karanjin is shown to possess pesticidal (Rangaswamy and Seshadri, 1941) and insecticidal (Parmar and Gulati, 1969) properties. Recently, antiinflammatory effect of karanjin in rat models as substantiated by the inhibition of lipoxygenase-1 and 5-LOX by karanjin (Sapna et al., 2007), has been reported. Also, anti-ulcer property of karanjin
Vismaya et al. / Industrial Crops and Products 32 (2010) 118–122
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by the inhibition of H+ , K+ -ATPase by in vitro studies using sheep parietal cells and in vivo using ulcer induced adult rat models has been established (Vismaya et al., 2008). Earlier work on the extraction, purification and isolation of karanjin indicates that it is a challenge to achieve its isolation in high purity due to its labile nature. Since the molecule possesses important biological functions, its isolation in pure form with high recovery would be a value addition to P. pinnata seed oil. Present investigation was aimed at standardization of process for the isolation of karanjin from karanja seed oil with an emphasis on achieving higher yields of pure karanjin and understanding the effect of processing methods on its structure.
Table 1 Karanjin content in seed, oil and cake of Pongamia pinnata.
2. Experimental
2.5. Characterization
2.1. Materials
2.5.1. Liquid chromatography and mass spectrometry LC–MS was carried out on Waters Spectrometer (Model Q-Tof Ultima). Electrospray ionization (ESI) with positive ionization technique was adopted to study karanjin.
Acetonitrile, methanol (HPLC-grade) and petroleum ether 40–60 ◦ C (AR Grade) were sourced from Fisher Scientific (Pittsburgh, PA, USA). Neutral alumina (70–230 mesh) was procured from Hi media laboratories, Mumbai, India. P. pinnata seeds were procured from M/s Suresh Forestry Network, Chickballapur, Karnataka, India. 2.2. Extraction Extraction of oil from karanja seeds (100 g) was carried out for 36 h using petroleum ether (500 mL) at room temperature followed by evaporation of solvent. The oil (33 g) was subjected to liquid–liquid extraction with methanol in the ratio of 1:2 (v/v). Extraction was repeated thrice for total of 96 h and the separated methanolic extracts were pooled (200 mL). This extract was concentrated and then subjected to chromatography for purification.
Source Whole seed Oil Ghani pressed cake Ghani pressed and hexane extracted cake Mean ± SD of triplicates.
relative percentage of karanjin were assessed based on the peak area.
2.5.2. NMR spectroscopy Purified karanjin sample (5.0 mg) was dissolved in 0.5 mL deuterated chloroform for NMR studies. 1 H spectra were recorded at 500 MHz on Bruker Avance 500 MHz spectrometer (Rheinstetten, Germany) in CDCl3 solvent (ı 7.24 ppm) as internal standard. 13 C spectra were recorded at 125 MHz with central peak of the CDCl3 triplet (ı 76.70 ppm) as internal standard. 1 H and 13 C spectral chemical shifts and coupling constants were expressed in ı and Hz, respectively. Pulse angle employed in DEPT experiments was 135◦ . Attached proton test was done by Spin Echo Fourier Transformation method on 13 C nuclei at 125 MHz. 1 H COSY experiment was performed to trace out through-bond proton–proton connectivity.
2.3. Purification
3. Results and discussions
The methanolic extract (3.8 g) was passed through neutral activated alumina column (20 cm × 1.5 cm). Elution was carried out using methanol. The eluent (400 mL) collected was free from pigments and other minor constituents. The solvent was distilled to afford a partially purified fraction. The recovery of karanjin from partially purified fraction was carried out by preparative HPLC (Shimadzu LC-8A, Shimadzu SPD-MIOA Diode array detector) using Silica column C5 (250 mm × 21.20 mm) with water–acetonitrile as solvent using linear gradient (20–80%; 110 min; flow rate, 4 mL/min). Purified karanjin was also obtained by taking the partially purified fraction (3 g) in 5 mL of acetonitrile:water (7:3, v/v) followed by crystallization at 7 ◦ C for 1 week. Pure karanjin thus obtained was used for characterization.
3.1. Assessment of karanjin
2.4. Quantification 2.4.1. Spectrophotometric method Quantitative assay of karanjin was made by measuring the absorbance at 259 nm and using molar extinction coefficient (log ∈ 4.56). This was done at various stages of isolation and expressed as karanjin equivalents in partially purified samples. 2.4.2. Chromatographic method The partially purified preparation and purified karanjin in methanol were analyzed by reverse phase analytical HPLC. HPLC was carried out using Waters 1525 model with UV detector 2996 (UV detection; = 259 nm) using an ODS column of C18 (150 mm × 4.1 mm) and eluted with a linear water–acetonitrile gradient (20–80%; 65 min; flow rate, 1 mL/min). The purity and
Karanjin (%) 1.90 ± 0.15 5.43 ± 0.20 0.53 ± 0.01 0.05 ± 0.00
Karanja seed contains inner kernel portion constituting around 94% and the remaining 6% forms the outer hull. The seed contained 1.9% of karanjin and it was present mainly in the oil (5.4%) as indicated in Table 1. However, during processing karanjin gets distributed (0.05–1.9 g/100 g material) in different products based on the amount of oil present indicating that karanjin originates from oil but not associated with any other macronutrients present in the karanja meal. Extraction of karanjin using different solvent systems showed variations in concentration based on the polarity of the solvents. Among these, methanol afforded the best results. The seed hull contained 6.6% karanjin whereas kernels had 2.0% (Table 2). Although the karanjin content was more pronounced in the hull portion, its contribution was less to the whole seed. Karanja seeds obtained from different regions were assessed for karanjin content, which was found to be ranging from 0.9 to 1.9%, probably due to the influence of environmental and agronomical conditions such as nature and fertility of soil. Seeds collected around Chickballapur region in Karnataka state, India used in the present study contained 1.9% karanjin and were found suitable for the extraction of karanjin.
Table 2 Oil, methanolic extractives and karanjin in P. pinnata seed components. Material
Oil (%)
Methanolic extractives (g/100 g oil)
Karanjin (%)
Whole seed Hull Kernal
33.1 ± 0.22 4.4 ± 0.20 39.2 ± 0.31
11.3 ± 0.15 37.6 ± 0.18 11.7 ± 0.12
1.9 ± 0.15 6.6 ± 0.30 2.0 ± 0.02
Mean ± SD of triplicates.
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Fig. 2. HPLC profile of purified karanjin.
ent stages, with a time consuming procedure and also the yield of karanjin was less (7%).
3.3. Evaluation of purity and recovery
Fig. 1. Flow chart for the isolation of karanjin from karanja seed oil along with material balance.
3.2. Extraction and isolation The recovery of karanjin provides value addition to P. pinnata seed oil which, at present, is being exploited for the production of bio-diesel. In the present study, optimization of a method for isolation of karanjin in pure form has been attempted with a view to increasing the yield and minimizing the processing steps (Fig. 1). It was observed that if karanjin was extracted directly from seeds, interference by other minor compounds would hinder the purification of karanjin during isolation. This was also reported by Simin et al. (2002). In our study, methanolic extract of karanja seed oil was taken up for the recovery of karanjin. Purification of karanjin by silica gel chromatography involves greater losses of karanjin resulting in poor yield (Roy et al., 1977; Simin et al., 2002). In the present study, losses of karanjin were minimized by using alumina for purification. Crystallization using acetonitrile-water mixture for isolation of karanjin was successful in obtaining over 20% yield. Isolation of karanjin has been described by Roy et al. (1977) during their studies on ‘Isopongaflavone’ from P. pinnata seeds. Though the steps involved in isolation were simple, the yield was less. Simin et al. (2002) isolated karanjin from karanja seeds while studying Pongarotene, a new rotenoid. The method involved the use of large number of solvents with a recovery of less than 0.5% of karanjin. The method of isolation of karanjin by Pathak et al. (1983) gave very poor yield (1%) of karanjin, during their study on ‘Isopongachromene’. Aneja et al. (1963) have reported the isolation of karanjin while studying the chromogenic factors present in the seed. It took long time for oil extraction involving two differ-
The purity of karanjin was evaluated by analytical HPLC. Purity was assessed in karanjin preparations before and after purification. After adsorption chromatography over alumina, the partially purified karanjin showed ∼80% purity. After adsorption chromatography, either preparative HPLC or crystallization was adopted for further purification. Karanjin obtained by crystallization in aqueous acetonitrile was found to be >95% pure (Fig. 2). Similarly, adsorption chromatography followed by preparative HPLC for purification gave >95% purity but with a poor recovery of 5%. The process of recovery through crystallization was very efficient in achieving a yield of karanjin up to 20%, which is much higher compared to earlier reported methods (0.5–7.0%). All methods reported so far have not addressed to the specific issue of high recovery and purity in isolation of karanjin. In the present method, this has been achieved by a single extraction step followed by purification with selected adsorbent and appropriate solvents. It was possible to achieve only a maximum of 20% recovery of karanjin, as losses during processing occur apparently due to its photo-labile nature. This is supported by Lakshmi et al. (1975), where conversion of karanjin to photo-karanjone has been reported.
Table 3 1 H (500 MHz) and karanjin. 1
␣  O-CH3 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6
13
C (125 MHz) spectral data (ı in ppm, d1-chloroform) for
H [ı, mult, J (Hz)]
7.78 d (2.0) 7.20 d (2.0) 3.85 s
8.22 d (8.5) 7.57 d (8.5)
8.17 m 7.60 m 7.60 m 7.60 m 8.17 m
13
C
145.4 103.9 59.9 154.5 141.5 174.7 121.6 109.7 157.8 116.7 149.6 130.7 119.4 128.0 128.3 130.3 128.3 128.0
HMBC [H → C] C-, C-7, C-8 C-␣, C-7, C-8 C-3
C-4, C-7, C-9 C-7, C-8, C-9, C-10
C-2, C-4 , C-6 C-1 , C-5 C-2 , C-6 C-1 , C-3 C-2, C-2 , C-4
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1H
COSY (Fig. 3) correlations between ␣ and  protons of furan ring, C-5 and C-6 protons of the aromatic ring and C2 , C-6 with that of C-3 , 5 protons, respectively. The compound was characterized as karanjin (Fig. 4). Mass spectra for the compound in the ESI+ mode exhibited peaks at 293 and 315 corresponding to (M+H+ ) and (M+Na+ ) adducts confirming the molecular weight of the molecule to be 292. 4. Conclusion A simple viable method has been standardized for isolation of karanjin from P. pinnata seed oil wherein higher yield of karanjin up to 20% with purity >95% was achieved. Confirmation of mass and structure of the molecule was accomplished by LC–MS and NMR spectroscopy. The studies revealed that the structure of karanjin was stable during processing steps adopted in the present method. Isolation of karanjin in pure form and high yield provides value addition to P. pinnata seed. Also, it would help pharmaceutical industry, for this molecule in pure form would be more useful in understanding of mechanism of its action instead of crude extract of the seed. Acknowledgements Fig. 3.
1
H COSY spectrum of karanjin.
3.4. Characterization 1 H Spectral data (Table 3) of pure karanjin, obtained in the present study, reveal the presence of characteristic signals for furan ring as ıH /ıC at ı 7.78 (d, J = 2 Hz, H␣ )/145.4 (C␣ ) and at ıH /ıC at ı 7.20 (d, J = 2 Hz, H )/103.9 (C ). Signals at 8.17 (2H, multiplet) and at 7.60 (3H, multiplet) indicated a mono-substituted aromatic ring. 13 C Spectral data (Table 3) showed the presence of a carbonyl carbon at 174.7 and a methoxyl carbon at 59.9. Two carbons at 145.4 and 157.8 and two more tertiary carbons at 149.6 and 154.5 indicate aromatic carbons linked to oxygen atom in a furano-flavonoid molecule. A mono-substituted aromatic ring is shown by signals at 130.3, 128.3 (two carbons) and 128.0 (two carbons). Also, the 13 C Spectrum for this compound contained four more signals for aromatic carbons at 121.6, 109.7, 119.4 and 116.7 along with a signal at 141.5 for the carbon attached to the methoxyl group. Also, HMBC [H → C] data are presented in Table 3. DEPT spectra showed signals for CH and CH3 carbons accounting for 10 carbons. Corresponding SEFT spectra showed positive peaks for these carbons and negative peaks for the other eight tertiary carbons. From the 1 H and 13 C spectral assignments along with DEPT and SEFT spectral data, it could be clearly inferred that annelation of the furan and flavanoid rings was at C7/C8 and C9/C10 positions, respectively and ring substitution at 2/1 position. Further, spectral assignments concurred with
Fig. 4. Structure of karanjin.
Sincere thanks are due to Dr. V. Prakash, Director, Central Food Technological Research Institute, Mysore for his constant support and encouragement during the course of investigation. We are thankful to Dr. A.G. Appu Rao, Head of Department of Protein Chemistry & Technology and Dr. B.R. Lokesh, Head, Lipid Science & Traditional Foods for their suggestions and advice during the study. We thank the Department of Biotechnology, Govt. of India for their financial support for the project. References Aneja, R., Khanna, R.N., Seshadri, T.R., 1963. 6-Methoxyfuroflavone, a new component of the seeds of Pongamia glabra. J. Chem. Soc., 163–168. Bringi, N.V., 1987. Non-traditional Oilseeds and Oils in India. Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi, India, pp. 143–166. CSIR, 1969. The Wealth of India—A dictionary of Indian Raw Materials. Vol 8. Publications and Information Directorate, Council of Scientific and Industrial Research, New Delhi, India, pp. 206–211. Dahanukumar, S.A., Kulkarni, R.A., Rege, N.N., 2000. Pharmacology of medicinal plants and natural products. Indian J. Pharmacol. 32, 81–118. De, B.K., Bhattacharya, D.K., 1999. Biodeisel from minor vegetable oils like karnaja oil and nahor oil. Fett Lipid. 101, 404–406. Elanchezhiyan, M., Rajarajan, S., Rajendran, P., Subramaniyan, S., Thyagarajan, S.P., 1993. Antiviral properties of the seed extract of an Indian medicinal plant Pongamia pinnata Linn., against herpes simplex virus: in vitro studies on Vero cells. J. Med. Microbiol. 38, 262–264. Lakshmi, P., Srimannarayana, G., Subba Rao, N.V., 1975. Behavior of karanjin under UV irradiation. Indian J. Chem. 13, 1094–1095. Maher, L.C., Vidya Dharmagadda, S.S., Naik, S.N., 2006. Optimization of alkalicatalyzed transesterification of Pongamia pinnata oil for production of bio-diesel. Bioresour. Technol. 97, 1392–1397. Muthu, C., Ayyanar, M., Raja, N., Ignacimuthu, S., 2006. Medicinal plants used by traditional healers in Kancheepuram district of Tamilnadu, India. J. Ethnobiol. Ethnomed. 2, 43. Parmar, B.S., Gulati, K.C., 1969. Synergists for pyrethrins (II)-karanjin. Indian J. Entomol. 31, 239–243. Pathak, V.P., Saini, T.R., Khanna, R.N., 1983. Isopongachromene, a chromenoflavone from Pongamia glabra seeds. Phytochemistry 22, 308–309. Raheman, H., Phadatare, A.G., 2004. Diesel engine emissions and performance from blends of karanja methyl ester and diesel. Biomass Bioenerg. 27, 393–397. Rangaswamy, S., Seshadri, T.R., 1941. Indian J. Pharmacol. 3, 3. Rao, N.V.S., Rao, J.V., 1941. A note on glabrin: a new component of the seeds of Pongamia glabra. Proc. Indian Acad. Sci. 14, 123–125. Roy, D., Sharma, N.N., Khanna, R.N., 1977. Structure and synthesis of isopongaflavone, a new component of the seeds of Pongamia glabra. Indian J. Chem. 15, 1138–1139. Sapna, W.E., Sindhu Kanya, T.C., Mamatha, A.M., Lokesh, B.R., Appu Rao, A.G., 2007. Karanjin, a flavonoid inhibits lipoxygenases. In: Proceedings of the National Academy of Science India, National symposium held during December 2007 at CFTRI, Mysore, India.
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Simin, K., Ali, Z., Uz Zaman, K., Viqar Uddin Ahmed, S.M., 2002. Structure and biological activity of a new rotenoid from Pongamia pinnata. Nat. Prod. Lett. 16, 351–357. Vinay, B.J., Sindhu Kanya, T.C., 2008. Effect of detoxification on functional and nutritional quality of proteins of karanja seed meal. Food Chem. 106, 77–84.
Vismaya, Sindhu, R., Srikanta, B.M., Shylaja, M.D., Sindhu Kanya, T.C., 2008. Anti-ulcer potential of karanjin: a furano-flavonoid from karanja seed oil. In: Proceedings of the Society of Biological Chemists (I), National symposium held during December 2008 at IIT(M), Chennai, India.
Contents lists available at ScienceDirect
Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Extraction and recovery of karanjin: A value addition to karanja (Pongamia pinnata) seed oil Vismaya a , W. Sapna Eipeson a , J.R. Manjunatha b , P. Srinivas b , T.C. Sindhu Kanya a,∗ a
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Council of Scientific & Industrial Research, Mysore 570020, India Department of Plantation Products, Spices and Flavour Technology, Central Food Technological Research Institute, Council of Scientific & Industrial Research, Mysore 570020, India b
a r t i c l e
i n f o
Article history: Received 29 August 2009 Received in revised form 21 March 2010 Accepted 30 March 2010
Keywords: Pongamia pinnata Karanja Karanjin Recovery Characterization NMR
a b s t r a c t Karanja (Pongamia pinnata) seed oil contains karanjin, a bioactive molecule with important biological attributes. The objective of this study was to develop a facile method for efficient recovery of karanjin. The seed oil was subjected to liquid–liquid extraction with methanol. The extract was further purified by chromatography on alumina followed by crystallization to afford karanjin, whose purity was ascertained by HPLC. The recovery of karanjin was ∼20% with >95% purity. The structure of the compound was elucidated by MS and NMR spectral analysis. Mass spectrum for the compound in ESI+ mode showed signals at 293 and 315 corresponding to (M+H+ ) and (M+Na+ ) adducts, respectively, confirming the molecular weight to be 292. 1 H and 13 C spectral data established the structure of the molecule to be a furanoflavonoid compound with the furan ring signals appearing at 7.78 (H␣ )/145.4 (C␣ ) and 7.20 (H )/103.9 (C ). 1 H COSY, HMBC (H → C) and DEPT spectral data showed the annelation of furan and flavonoid rings to be at C7/C8 and C9/C10 positions, respectively, and a phenyl ring substitution at 2 (1 ) position. Karanjin’s recovery in pure form and high yield affords value addition to the seed oil for pharmaceutical applications. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Many unconventional oilseeds from forest produce have been explored to overcome the shortage and high price of oils from traditional sources. They find several uses and one such seed is from Pongamia pinnata (L.) Pierre (Leguminaceae, Papilionaceae; synonym Pongamia glabra Vent.), popularly known as ‘Karanj’ or ‘Karanja’, which has received greater attention (CSIR, 1969). This medium-size tree is indigenous to the Indian subcontinent and south-east Asia. It is one of the widely grown forest trees with 0.11 million tons of seeds collected every year in India, by states like Andhra Pradesh, Karnataka and Tamil Nadu (Vinay and Sindhu Kanya, 2008). Historically, this plant has long been used in India and neighboring regions as a source of traditional medicine, animal fodder, green manure, timber and fuel. Karanja seed containing 33–36% oil is also a rich source of protein (20–28%). The oil from P. pinnata has the potential to provide an environmentally acceptable, alternative to conventional diesel engine fuels (Raheman and Phadatare, 2004). At present, karanja oil is being explored mainly for its use as bio-diesel (De and Bhattacharya, 1999; Maher et al., 2006). The de-oiled cake of P. pinnata seeds obtained after solvent
∗ Corresponding author. Tel.: +91 821 2515331; fax: +91 821 2517233. E-mail address: [email protected] (T.C. Sindhu Kanya). 0926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2010.03.011
extraction contains up to 30% protein. While it has the potential to be used as sustainable animal feed supplement, its anti-nutritional factors restrict its usage as a feed/food. Indeed, nutritional quality of karanja seed proteins was improved after reducing the antinutrients in the meal (Vinay and Sindhu Kanya, 2008). Karanja seed is used as a medicinal plant, particularly with the Ayurvedic and Sidda medicine systems of India (Muthu et al., 2006). Crude seed extract can completely inhibit the growth of herpes simplex virus type 1 and type 2 in Vero cells (Elanchezhiyan et al., 1993) and also possesses hypoglycemic, anti-oxidative, anti-ulcerogenic, anti-inflammatory and analgesic properties (Dahanukumar et al., 2000). Karanja oil contains 5–6% flavonoids (Bringi, 1987), the main constituents being karanjin, a furano-flavonoid and pongamol, a diketone. Karanjin has been isolated during the course of exploration of newer compounds from karanja seeds. Earlier works of Rao and Rao (1941) have led several workers (Aneja et al., 1963; Roy et al., 1977; Pathak et al., 1983), to identify some new components of Pongamia seed oil apart from karanjin. Many biological activities of P. pinnata seed extracts can be attributed to karanjin, the major flavonoid of the seed oil. Karanjin is shown to possess pesticidal (Rangaswamy and Seshadri, 1941) and insecticidal (Parmar and Gulati, 1969) properties. Recently, antiinflammatory effect of karanjin in rat models as substantiated by the inhibition of lipoxygenase-1 and 5-LOX by karanjin (Sapna et al., 2007), has been reported. Also, anti-ulcer property of karanjin
Vismaya et al. / Industrial Crops and Products 32 (2010) 118–122
119
by the inhibition of H+ , K+ -ATPase by in vitro studies using sheep parietal cells and in vivo using ulcer induced adult rat models has been established (Vismaya et al., 2008). Earlier work on the extraction, purification and isolation of karanjin indicates that it is a challenge to achieve its isolation in high purity due to its labile nature. Since the molecule possesses important biological functions, its isolation in pure form with high recovery would be a value addition to P. pinnata seed oil. Present investigation was aimed at standardization of process for the isolation of karanjin from karanja seed oil with an emphasis on achieving higher yields of pure karanjin and understanding the effect of processing methods on its structure.
Table 1 Karanjin content in seed, oil and cake of Pongamia pinnata.
2. Experimental
2.5. Characterization
2.1. Materials
2.5.1. Liquid chromatography and mass spectrometry LC–MS was carried out on Waters Spectrometer (Model Q-Tof Ultima). Electrospray ionization (ESI) with positive ionization technique was adopted to study karanjin.
Acetonitrile, methanol (HPLC-grade) and petroleum ether 40–60 ◦ C (AR Grade) were sourced from Fisher Scientific (Pittsburgh, PA, USA). Neutral alumina (70–230 mesh) was procured from Hi media laboratories, Mumbai, India. P. pinnata seeds were procured from M/s Suresh Forestry Network, Chickballapur, Karnataka, India. 2.2. Extraction Extraction of oil from karanja seeds (100 g) was carried out for 36 h using petroleum ether (500 mL) at room temperature followed by evaporation of solvent. The oil (33 g) was subjected to liquid–liquid extraction with methanol in the ratio of 1:2 (v/v). Extraction was repeated thrice for total of 96 h and the separated methanolic extracts were pooled (200 mL). This extract was concentrated and then subjected to chromatography for purification.
Source Whole seed Oil Ghani pressed cake Ghani pressed and hexane extracted cake Mean ± SD of triplicates.
relative percentage of karanjin were assessed based on the peak area.
2.5.2. NMR spectroscopy Purified karanjin sample (5.0 mg) was dissolved in 0.5 mL deuterated chloroform for NMR studies. 1 H spectra were recorded at 500 MHz on Bruker Avance 500 MHz spectrometer (Rheinstetten, Germany) in CDCl3 solvent (ı 7.24 ppm) as internal standard. 13 C spectra were recorded at 125 MHz with central peak of the CDCl3 triplet (ı 76.70 ppm) as internal standard. 1 H and 13 C spectral chemical shifts and coupling constants were expressed in ı and Hz, respectively. Pulse angle employed in DEPT experiments was 135◦ . Attached proton test was done by Spin Echo Fourier Transformation method on 13 C nuclei at 125 MHz. 1 H COSY experiment was performed to trace out through-bond proton–proton connectivity.
2.3. Purification
3. Results and discussions
The methanolic extract (3.8 g) was passed through neutral activated alumina column (20 cm × 1.5 cm). Elution was carried out using methanol. The eluent (400 mL) collected was free from pigments and other minor constituents. The solvent was distilled to afford a partially purified fraction. The recovery of karanjin from partially purified fraction was carried out by preparative HPLC (Shimadzu LC-8A, Shimadzu SPD-MIOA Diode array detector) using Silica column C5 (250 mm × 21.20 mm) with water–acetonitrile as solvent using linear gradient (20–80%; 110 min; flow rate, 4 mL/min). Purified karanjin was also obtained by taking the partially purified fraction (3 g) in 5 mL of acetonitrile:water (7:3, v/v) followed by crystallization at 7 ◦ C for 1 week. Pure karanjin thus obtained was used for characterization.
3.1. Assessment of karanjin
2.4. Quantification 2.4.1. Spectrophotometric method Quantitative assay of karanjin was made by measuring the absorbance at 259 nm and using molar extinction coefficient (log ∈ 4.56). This was done at various stages of isolation and expressed as karanjin equivalents in partially purified samples. 2.4.2. Chromatographic method The partially purified preparation and purified karanjin in methanol were analyzed by reverse phase analytical HPLC. HPLC was carried out using Waters 1525 model with UV detector 2996 (UV detection; = 259 nm) using an ODS column of C18 (150 mm × 4.1 mm) and eluted with a linear water–acetonitrile gradient (20–80%; 65 min; flow rate, 1 mL/min). The purity and
Karanjin (%) 1.90 ± 0.15 5.43 ± 0.20 0.53 ± 0.01 0.05 ± 0.00
Karanja seed contains inner kernel portion constituting around 94% and the remaining 6% forms the outer hull. The seed contained 1.9% of karanjin and it was present mainly in the oil (5.4%) as indicated in Table 1. However, during processing karanjin gets distributed (0.05–1.9 g/100 g material) in different products based on the amount of oil present indicating that karanjin originates from oil but not associated with any other macronutrients present in the karanja meal. Extraction of karanjin using different solvent systems showed variations in concentration based on the polarity of the solvents. Among these, methanol afforded the best results. The seed hull contained 6.6% karanjin whereas kernels had 2.0% (Table 2). Although the karanjin content was more pronounced in the hull portion, its contribution was less to the whole seed. Karanja seeds obtained from different regions were assessed for karanjin content, which was found to be ranging from 0.9 to 1.9%, probably due to the influence of environmental and agronomical conditions such as nature and fertility of soil. Seeds collected around Chickballapur region in Karnataka state, India used in the present study contained 1.9% karanjin and were found suitable for the extraction of karanjin.
Table 2 Oil, methanolic extractives and karanjin in P. pinnata seed components. Material
Oil (%)
Methanolic extractives (g/100 g oil)
Karanjin (%)
Whole seed Hull Kernal
33.1 ± 0.22 4.4 ± 0.20 39.2 ± 0.31
11.3 ± 0.15 37.6 ± 0.18 11.7 ± 0.12
1.9 ± 0.15 6.6 ± 0.30 2.0 ± 0.02
Mean ± SD of triplicates.
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Fig. 2. HPLC profile of purified karanjin.
ent stages, with a time consuming procedure and also the yield of karanjin was less (7%).
3.3. Evaluation of purity and recovery
Fig. 1. Flow chart for the isolation of karanjin from karanja seed oil along with material balance.
3.2. Extraction and isolation The recovery of karanjin provides value addition to P. pinnata seed oil which, at present, is being exploited for the production of bio-diesel. In the present study, optimization of a method for isolation of karanjin in pure form has been attempted with a view to increasing the yield and minimizing the processing steps (Fig. 1). It was observed that if karanjin was extracted directly from seeds, interference by other minor compounds would hinder the purification of karanjin during isolation. This was also reported by Simin et al. (2002). In our study, methanolic extract of karanja seed oil was taken up for the recovery of karanjin. Purification of karanjin by silica gel chromatography involves greater losses of karanjin resulting in poor yield (Roy et al., 1977; Simin et al., 2002). In the present study, losses of karanjin were minimized by using alumina for purification. Crystallization using acetonitrile-water mixture for isolation of karanjin was successful in obtaining over 20% yield. Isolation of karanjin has been described by Roy et al. (1977) during their studies on ‘Isopongaflavone’ from P. pinnata seeds. Though the steps involved in isolation were simple, the yield was less. Simin et al. (2002) isolated karanjin from karanja seeds while studying Pongarotene, a new rotenoid. The method involved the use of large number of solvents with a recovery of less than 0.5% of karanjin. The method of isolation of karanjin by Pathak et al. (1983) gave very poor yield (1%) of karanjin, during their study on ‘Isopongachromene’. Aneja et al. (1963) have reported the isolation of karanjin while studying the chromogenic factors present in the seed. It took long time for oil extraction involving two differ-
The purity of karanjin was evaluated by analytical HPLC. Purity was assessed in karanjin preparations before and after purification. After adsorption chromatography over alumina, the partially purified karanjin showed ∼80% purity. After adsorption chromatography, either preparative HPLC or crystallization was adopted for further purification. Karanjin obtained by crystallization in aqueous acetonitrile was found to be >95% pure (Fig. 2). Similarly, adsorption chromatography followed by preparative HPLC for purification gave >95% purity but with a poor recovery of 5%. The process of recovery through crystallization was very efficient in achieving a yield of karanjin up to 20%, which is much higher compared to earlier reported methods (0.5–7.0%). All methods reported so far have not addressed to the specific issue of high recovery and purity in isolation of karanjin. In the present method, this has been achieved by a single extraction step followed by purification with selected adsorbent and appropriate solvents. It was possible to achieve only a maximum of 20% recovery of karanjin, as losses during processing occur apparently due to its photo-labile nature. This is supported by Lakshmi et al. (1975), where conversion of karanjin to photo-karanjone has been reported.
Table 3 1 H (500 MHz) and karanjin. 1
␣  O-CH3 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6
13
C (125 MHz) spectral data (ı in ppm, d1-chloroform) for
H [ı, mult, J (Hz)]
7.78 d (2.0) 7.20 d (2.0) 3.85 s
8.22 d (8.5) 7.57 d (8.5)
8.17 m 7.60 m 7.60 m 7.60 m 8.17 m
13
C
145.4 103.9 59.9 154.5 141.5 174.7 121.6 109.7 157.8 116.7 149.6 130.7 119.4 128.0 128.3 130.3 128.3 128.0
HMBC [H → C] C-, C-7, C-8 C-␣, C-7, C-8 C-3
C-4, C-7, C-9 C-7, C-8, C-9, C-10
C-2, C-4 , C-6 C-1 , C-5 C-2 , C-6 C-1 , C-3 C-2, C-2 , C-4
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1H
COSY (Fig. 3) correlations between ␣ and  protons of furan ring, C-5 and C-6 protons of the aromatic ring and C2 , C-6 with that of C-3 , 5 protons, respectively. The compound was characterized as karanjin (Fig. 4). Mass spectra for the compound in the ESI+ mode exhibited peaks at 293 and 315 corresponding to (M+H+ ) and (M+Na+ ) adducts confirming the molecular weight of the molecule to be 292. 4. Conclusion A simple viable method has been standardized for isolation of karanjin from P. pinnata seed oil wherein higher yield of karanjin up to 20% with purity >95% was achieved. Confirmation of mass and structure of the molecule was accomplished by LC–MS and NMR spectroscopy. The studies revealed that the structure of karanjin was stable during processing steps adopted in the present method. Isolation of karanjin in pure form and high yield provides value addition to P. pinnata seed. Also, it would help pharmaceutical industry, for this molecule in pure form would be more useful in understanding of mechanism of its action instead of crude extract of the seed. Acknowledgements Fig. 3.
1
H COSY spectrum of karanjin.
3.4. Characterization 1 H Spectral data (Table 3) of pure karanjin, obtained in the present study, reveal the presence of characteristic signals for furan ring as ıH /ıC at ı 7.78 (d, J = 2 Hz, H␣ )/145.4 (C␣ ) and at ıH /ıC at ı 7.20 (d, J = 2 Hz, H )/103.9 (C ). Signals at 8.17 (2H, multiplet) and at 7.60 (3H, multiplet) indicated a mono-substituted aromatic ring. 13 C Spectral data (Table 3) showed the presence of a carbonyl carbon at 174.7 and a methoxyl carbon at 59.9. Two carbons at 145.4 and 157.8 and two more tertiary carbons at 149.6 and 154.5 indicate aromatic carbons linked to oxygen atom in a furano-flavonoid molecule. A mono-substituted aromatic ring is shown by signals at 130.3, 128.3 (two carbons) and 128.0 (two carbons). Also, the 13 C Spectrum for this compound contained four more signals for aromatic carbons at 121.6, 109.7, 119.4 and 116.7 along with a signal at 141.5 for the carbon attached to the methoxyl group. Also, HMBC [H → C] data are presented in Table 3. DEPT spectra showed signals for CH and CH3 carbons accounting for 10 carbons. Corresponding SEFT spectra showed positive peaks for these carbons and negative peaks for the other eight tertiary carbons. From the 1 H and 13 C spectral assignments along with DEPT and SEFT spectral data, it could be clearly inferred that annelation of the furan and flavanoid rings was at C7/C8 and C9/C10 positions, respectively and ring substitution at 2/1 position. Further, spectral assignments concurred with
Fig. 4. Structure of karanjin.
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