Quantication of water soluble vitamins in six date palm
(Phoenix dactylifera L.) cultivars fruits growing in Dubai, United Arab Emirates, through high performance liquid chromatography Junaid Aslam a,c, * , Sheba Haque Khan b , Saeed Ahmad Khan a a Dubai Pharmacy College, Al Muhasinah, P.O. Box 19099, Dubai, United Arab Emirates b G.F. College, Department of Chemistry, M.J.P. Rohilkhand University, Bareilly, Uttar Pradesh, India c Department of Biotechnology, Jamia Hamdard (Hamdard University), New Delhi 110062, India Received 19 January 2011; accepted 21 February 2011 Available online 24 February 2011 KEYWORDS Arecaceae; Date palm (Phoenix dactylifera L.); Water soluble vitamins; High performance liquid chromatography Abstract In present investigation, a comparative analysis of water soluble vitamins viz., B1 (thia- mine HCl), B2 (riboavin), B3 (nicotinamide), B5 (pantothenic acid), B6 (pyridoxine HCl), B9 (folic acid), B12 (cyanocobalamin) was carried out in fruits (immature, semimature and mature) of six date palm (Phoenix dactylifera L.) cultivars (Barhee, Khalasah, Muzati, Shishi, Zart, Zardai) growing in United Arab Emirates (UAE) by high performance liquid chromatograph (HPLC). The fruits were collected at three developing stages (immature, semimature and mature). Quantitative analysis of water soluble vitamins yield showed a signicant variation within the dif- ferent cultivars and the developing stages of date palm fruit. Vitamin B1, B3, B5, B6 were maximum (lg/100 g f.w.) in Shishi, Zardai, Shishi and Muzati at their matured stage, however, vita- min B2, B9, B12 were detected in immature fruit of Khalasah, Khalasah and Shishi culti- vars. The vitamin production in fruits of different date palm cultivars was, therefore, developing * Corresponding author at: Department of Biotechnology, Jamia Hamdard (Hamdard University), New Delhi 110062, India. Tel.: +91 99907 06557. E-mail address: Junaidg1@gmail.com (J. Aslam). 1319-6103 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of King Saud University. doi:10.1016/j.jscs.2011.02.015 Production and hosting by Elsevier Journal of Saudi Chemical Society (2013) 17, 916 King Saud University Journal of Saudi Chemical Society www.ksu.edu.sa www.sciencedirect.com stage specic and cultivar dependent. The present study showed that the date palm fruit could be used for human consumption with value addition of water soluble vitamins at their specic devel- opmental stages. 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. 1. Introduction The date palm (Phoenix dactilyfera L.), species has 19 known genetic relatives. The genus Phoenix belongs to the family Arecaceae. The fruit is a Berry type (known also as Drupe) with a single seed in each. Date palm is one of the most impor- tant cash crops; it is especially grown in Arab region and the area bordering the Mediterranean coast. It has been cultivated since the prehistoric time in most of the Gulf, Asian and Afri- can countries. More than 2000 date palm cultivars enlisted through the world, but few of them have been screened for their large agronomic performance and fruit quality (Al-Hooti et al., 1997). Due to the nutritional value of the fruit; research- ers have focused their attention on characterization of the physico-chemical analysis of the important date palm cultivars (Al-Shahib and Marshall, 2002; Mrabet et al., 2008). Increase in date fruit production will, therefore, play a signicant role worldwide in the improvement of nutritional status of people, with special reference to calories and important minerals (Ahmed et al., 1995). It is usually taken raw or with milk, which makes it highly nutritious in most respects. Taking it with curdled milk is also common. The seeds of the fruit, roasted and ground into powder makes a beverage like coffee, called date coffee. The date palm yields a sweet juice of high food value. It can be taken fresh with great advantage or made into a tasty country sugar. The juice can also be allowed to fer- ment and made into alcohol. It provides natural sugar in the form of glucose and fructose. This sugar is ready for immedi- ate absorption and is, therefore, innitely superior to cane- sugar. Dates are rich in sugar ranging from 65% to 80% on dry weight basis mostly of inverted form (glucose and fruc- tose). Fresh varieties have a higher content of inverted sugars; semi dried varieties contain equal amounts of inverted and sucrose, while dried varieties contain higher sucrose. Water content is between 7% (dried) and 79% (fresh) depending on the cultivars (Myhara et al., 1999; Al-Farsi et al., 2005). Therefore, the moisture, total nitrogen, fat, ber, ash, tannins, vitamin C, b-carotene and ten nutritionally important minerals were highest in the early stage of development and decreased during maturation. Date fruit, being exceptionally rich in potassium and extremely low in sodium (Ahmed et al., 1995). Dates are valuable as medicine for their tonic effect. Being easily digested, they are very useful for supplying energy and repairing waste. Milk in which fresh and cleaned dates have been boiled proved very nourishing and proved as a restorative drink for children and adults alike, especially during convales- cence. Anumber of studies have been carried out to characterize its chemical composition particularly minerals, polysaccha- rides, carbohydrates in fresh date fruit (Fayadah and Al Showiman, 1990; Al-Hooti et al., 1997;Al-Shahib and Marshall, 2002). It has been reported that the vitamins play a potent role to reduce the damage carried out by the free radicals and check degenerative disease (Jacab and Sotoudeh, 2002). In addition, vitamin supplements showed the ergogenic and performance enhancing effects (Clarkson, 1993; Zhao et al., 2004). Thus, chemical and physical characterization has been carried out pre- viously by many workers (Al-Juburi et al., 1994; Al-Hooti et al., 1997; Attalla and Harraz, 1996; Mrabet et al., 2008), but there is a distinct lack of information on water soluble vitamins during the ripening stages of date palm fruit. Therefore, the present investigation was undertaken to determine the presence of water-soluble vitamins (Fig. 1) in fruits of selected date palm cultivars at their developmental stages (immature, semimature and mature) growing in Dubai, United Arab Emirates. 2. Experimental 2.1. Experimental material The fruits of six different date palm (Phoenix dactylifera L.) cultivars (Barhee, Khalasah, Muzati, Shishi, Zart, Zardai) were collected at their developing stages (immature, semimature and mature) from Al-Muhasinah-1, Dubai, Uni- ted Arab Emirates. 2.2. Reagents and solvents The reagents and chemicals were used in the present study were of analytical grade viz., HPLC methanol (Merck), aceto- nitrile HPLC (Merck), glacial acetic acid (BDH), triethylamine (RDH), orthophosphoric acid (BDH), pic6 (hexane sulfonic acid), sodium salt (Merck). The Millipore direct q system based distilled and deionized water was used for the prepara- tion of reagents and stock solution. 2.3. Standard preparation The standard stock solution of the water soluble vitamins was prepared as follows: (a) vitamin B1 (thiamine HCl) was pre- pared by dissolving 26.7 mg of thiamine hydrochloride in 25 ml of double distilled water; (b) vitamin B2 (riboavin) was prepared by dissolving 6.9 mg of riboavin in 100 ml of extraction solution (the extraction solution has limit to dis- solve 7 mg of riboavin); (c) vitamin B3 (nicotinamide) was prepared by dissolving 41.5 mg of nicotinamide in 25 ml of double distilled water; (d) vitamin B5 (calcium salt of panto- thenic acid) was prepared by dissolving 21.4 mg of calcium d-pantothenate in 25 ml of double distilled water; (e) vitamin B6 (pyridoxine HCl) was prepared by dissolving 20.8 mg of pyridoxine hydrochloride in 25 ml of double distilled water; (f) vitamin B9 (folic acid) was prepared by dissolving 11.2 mg of folic acid in 25 ml of double distilled water; (g) vita- min B12 (cyanocobalamin) was prepared by dissolving 19.8 mg of cyanocobalamin in 25 mg of double distilled water. 2.4. Working standard The working standards were prepared as follows: (i) B1, 1.068 mg/ml and 562 ll was taken in 10 ml volumetric ask, 10 J. Aslam et al. (ii) B2, 0.069 mg/ml and 5.1 ml was taken in 10 ml volumetric ask, (iii) B3, 1.66 mg/ml and 60.5 ll was taken in 10 ml volu- metric ask, (iv) B5, 0.856 mg/ml and 292 ll was taken in 10 ml volumetric ask, (v) B6, standard 0.832 mg/ml and 72.2 ll was taken in 10 ml volumetric ask, (vi) B9, standard 0.072 mg/ml and 132 ll was taken in 10 ml volumetric ask, (vii) and then B12, standard, 1.23 mg/ml and 142 ll was taken in 10 ml volumetric ask and then all were nally mixed. 2.5. Preparation of buffer, solutions and samples The buffer and sample preparation was carried out according to our previous report (Junaid et al., 2008). In short, 1.08 g of hexane sulfonic acid sodium salt and 1.36 g of potassium dihy- drogen phosphate were dissolved in 940 ml of HPLC water and 5 ml of triethylamine was added to it and the pH was ad- justed to 3.0 with orthophosphoric acid. To prepare the mobile phase, buffer and methanol were mixed with a ratio of 96:4 and ltered through 0.45 l membrane lter and degassed by using helium gas. Extraction solution was made by mixing 50 ml of acetonitrile with 10 ml of glacial acetic acid and the volume was nally made up to 1000 ml with double distilled water. Ten grams of each sample was homogenized, weighed and transferred into conical asks and 25 ml of extraction solution was added, water bath was kept shaking at 70 C for 40 min. Thereafter, the sample was cooled down, ltered and nally the volume was made up to 50 ml with extraction solution. 2.6. High performance liquid chromatography (HPLC) The ve point calibration curve was prepared using mix stan- dard in mobile phase. It was analyzed independently using HPLC and a standard curve was plotted between the concen- tration and the peak area. The injected quantities of the mix standard showed a good linearity. The data of peak area vs. the used standard vitamin concentrations were treated by lin- ear least-square regression and the regression equation thus Figure 1 Chemical structure of water soluble vitamins. Quantication of water soluble vitamins in six date palm (Phoenix dactylifera L.) cultivars fruits 11 obtained from standard curve was used to quantify the water- soluble vitamins in different date palm fruit samples. The col- umn used for the analysis was waters symmetry C18 (4.6 250 mm 5 lm) with a linear gradient of buffer/methanol (96:4) at a constant ow rate of 1 ml/min with 1800 pressure by using waters pump (1515 isocratic). A UV (2487) detector was employed for the detection of peaks, using two channels simul- taneously at a wavelength of 210 nm, a bandwidth of 5 nm and another wavelength of 280 nm. 2.7. Recovery study The accuracy of the current method was conrmed in the sam- ple using the recovery study. Pre-analyzed samples were spiked with extra 50%, 100% and 150% of the standard vitamins; the mixture of the standard was reanalyzed by adopting the pro- posed method. The experiment was repeated four times and the average recovered vitamins content was quantied using regression equation, and the % recovery was calculated accordingly. 2.8. Precision The precision of the used method was obtained by determining inter and intra-day variations in ve replicates of water-soluble vitamins at two concentration levels (200400 ng per spot) and the percent standard relative deviation (RSD) was calculated accordingly. 2.9. Quantication of water soluble vitamins Five replicates of each sample were analyzed using 60 ll. The yield was calculated using regression equation of calibration curve of each standard. 2.10. Statistic analysis The signicant and non-signicant variations within water sol- uble vitamin contents and the six date palm cultivars fruits were analyzed using one-way analysis of variance (ANOVAs). Values are means of ve replicates from two experiments, and the presented mean values were separated using Duncans Multiple Range Test (DMRT) at p 6 0.05. 3. Results and discussion In the present study, water soluble vitamins viz., B1 (thiamine HCl), B2 (riboavin), B3 (nicotinamide), B5 (pantothenic acid), B6 (pyridoxine HCl), B9 (folic acid), B12 (cyanocobala- min) were analyzed in fruits (immature, semimature, mature) of six date palm (Phoenix dactylifera L.) cultivars (Barhee, Khalasah, Muzati, Shishi, Zart, Zardai) growing (Fig. 2) in Dubai, United Arab Emirates. The solvent system (buffer/methanol, 96:4) used for the analysis produced a sharp and compact peak of the studied vitamins. The densitometric analysis of water soluble vitamins was carried out at 210 and 280 nm absorbance mode, and the regression analysis data showed a good linear relationship (Table 1). The method used for the vitamin extraction and subsequent estimation afforded recovery of 99.05100.11%. The inter- and intra-variation of vitamins at two different con- centration levels showed a low relative standard deviation (0.901.02%). The yield of the water soluble vitamins in differ- ent samples was analyzed from the regression equation using value of area obtained from win-cat software. Of the screened six date palm cultivars fruits; thiamine (B1) was only detected in Khalasah, Shishi and Muzati. In khalasah and Muzati cultivars, it was detected only in semimature and mature fruit; however, in Shishi, it was present in all the developmental stages of fruit. The maximum thiamine content was noticed in Khalasah followed by Shi- shi at mature level, thereafter; it started to decrease in semi- mature and mature fruits. In Barhee, Zart and Zardai; thiamine content was altogether absent (Fig. 3A). Figure 2 Chromatogram showing baseline separation of water soluble vitamins from date palm fruit samples: (1) cyanocobal- amin (B12), (2) nicotinamide (B3), (3) pantothenic acid (B5), (4) pyridoxine (B6), (5) thiamine (B1), (6) folic acid (B9), (7) riboavin (B2). Table 1 The important parameters of calibration curve. Water soluble vitamins y = ax + b r 2 Concentration range (mg/ml) Recovery Precision (R.S.D. (%)) Detection limit (mg l 1 ) B1 Y = 1.23e + 005x 1.17e + 004 0.9947 1.8620.8 0.70 2.43 0.1 B2 Y = 6.08e + 004x 4.59e + 005 0.9987 1.56320.01 0.62 3.08 0.2 B3 Y = 1.6e + 005x 2.37e + 004 0.9992 4.2052.0 0.78 3.64 0.5 B5 Y = 7.50e + 004x 1.13e + 005 0.9963 4.6950.4 1.2 5.58 0.1 B6 Y = 2.75e + 005x + 1.2e + 004 0.9921 1.5420.04 0.66 3.29 0.5 B9 Y = 1.31e + 005x 1.39e + 004 0.9987 2.5450.5 1.24 4.88 0.2 B12 Y = 5.07e + 004x 3.51e + 005 0.9945 1.8720.4 0.90 4.38 0.1 a slope; b intercept; r correlation coefcient. 12 J. Aslam et al. 0 2 4 6 8 10 12 14 Khalasah Shishi Muzati Barhee Zart Zardai Cultivars T h i a m i n e
u g / 1 0 0
g
f . w . Immature Semimature Mature a b c a b c a d d b d d b d d b b b (A) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Khalasah Shishi Muzati Barhee Zart Zardai Cultivars R i b o f l a v i n
u g / 1 0 0
g
f . w . b a a c b a c a a d c a c a d a c d (B) 0 5 10 15 20 25 30 35 40 45 50 Khalasah Shishi Muzati Barhee Zart Zardai Cultivars N i c o t i n a m i d e
u g / 1 0 0
g
f . w . a b b c c a a b c c a a b c d d d d (C) 0 1 2 3 4 5 6 7 8 9 Khalasah Shishi Muzati Barhee Zart Zardai Cultivars P a n t o t h e n i c
a c i d
u g / 1 0 0 g
f . w . a b a b a b c c b c c b c c b c c (D) Figure 3 HPLC analysis of water soluble vitamins in fruits of different date palm cultivars (A) thiamine (B1), (B) riboavin (B2), (C) nicotinamide (B3), (D) pantothenic acid (B5), (E) pyridoxine (B6), (F) folic acid (B9), (G) cyanocobalamin (B12). Quantication of water soluble vitamins in six date palm (Phoenix dactylifera L.) cultivars fruits 13 A signicant variation in riboavin content was noticed amongst the tested date palm fruits. The riboavin content was detected only in Khalasah, Shishi and Barhee fruits. In Khalasah and Shishi, it was isolated from semi- mature and mature fruit; however, in Barhee, it was quanti- ed only in semimature fruit. Among the Khalasah, Shishi, and Barhee; the highest riboavin content was quantied in Khalashs immature fruit followed by Barhee. Riboavin was not detected in Muzati, Zart and Zardai cultivars. Fig. 3B showed a comparative account of the various level of the riboavin content at different fruit developmental stages. Nicotinamide (B3) was detected in Khalash, Muzati, Barhee, Zart and Zardai cultivars. In Muzati, Bar- hee, Zart and Zardai, nicotinamide content was isolated in all the fruit developing stages; however, in Khalasah it analyzed only in semimature and mature fruit. The maximum vitamin content was noticed in mature fruit of Zardai culti- var; hence, no signicant difference (p 6 0.05) was noticed in Barhee, Zart and Zardai cultivars. Similarly, no signif- icant variation in nicotinamide yield was noticed in semima- ture and mature fruits of Khalasah and Muzati cultivars. In Shishi, it was altogether absent. The maximum yield of nicotinamide was detected in mature fruit of Zardai followed by Barhee and Zart cultivars (Fig. 3C). The se- quence of nicotinamide production at different developing stages of fruit was as follows: mature, semimatute and immature. Pantothenic acid was detected only in two cultivars: (a) Shishi and (b) Muzati. Although, in Muzati, it was iso- lated only in the semi mature and mature fruits, while in the immature, no signicant difference in the yield was, however, 0 0.5 1 1.5 2 2.5 3 3.5 4 Khalasah Shishi Muzati Barhee Zart Zardai Cultivars P y r i d o x i n e
u g / 1 0 0 g
f . w . a b c d d a b c a b c b b b d (E) e e c 0 0.5 1 1.5 2 2.5 3 3.5 4 Khalasah Shishi Muzati Barhee Zart Zardai Cultivars F o l i c
a c i d
u g / 1 0 0 g
f . w . a b a a b b c c c c c c c c c c c c (F) 0 0.5 1 1.5 2 2.5 Khalasah Shishi Muzati Barhee Zart Zardai Cultivars C y n o c o b a l a m i n
u g / 1 0 0
g
f . w . a b a b b b ab cd d d (G) c c c e c c c c Figure 3 (continued) 14 J. Aslam et al. noticed. The highest production was noticed in the mature fruit of Shishi cultivar. Fig. 3D showed pantothenic acid production at different developing stages of date palm fruit. Pyridoxine was quantied in most of the cultivars except Barhee, whereas, the highest content was noticed in imma- ture followed by semimature and mature fruits. The maximum pyridoxine content was detected in immature Muzati fruit followed by Shishi, Khalasah, Zart and Zardai culti- vars. Similarly, in semimature fruits, the highest vitamin con- tent was isolated from Muzati followed by Shishi, Khalasah, Zardai and Zart. Hence, no signicant dif- ference in pyridoxine content was detected in Khalasah and Zardai semimature fruits. In mature fruits, pyridoxine was also detected, but was in very low concentration. The highest vitamin yield was noticed in Muzati mature fruit. No signicant difference was observed in Khalasah, Shishi, Zart and Zardai cultivar fruits (Fig. 3E). In Barhee, it was altogether absent in all the tested fruit types. Folic acid was detected only in Khalasah and Zardai cultivars; hence, a signicant variation in content was noticed. The maximum folic acid was detected in immature fruit of Khalasah and semimature fruit of Zardai cultivars. It was completely absent in Shishi, Muzati, Barhee and Zart cultivars. Fig. 3F showed a comparative account of the folic acid production at different developing stages of fruit. The analysis of cyanocobalamin showed that it was present in all the developing stages of Khalasah and Shishi fruits; however, in Muzati it was detected only in semimature and mature fruits. In Zart and Zardai, it was noticed only in mature fruits. The highest yield of cyanocobalamin was detected in the immature fruit of Shishi followed by Khalasah. No signicant difference was found in the semi- mature fruits of Khalasah and Muzati. Similarly, in mature fruits of Khalasah and Shishi no signicant vari- ation was noticed in yield. In addition, the content isolated from mature fruits of Muzati, Zart and Zardai was not signicantly different. Moreover, the cyanocobalamin con- tent in immature and mature fruits of Barhee, Zart and Zardai was altogether absent. Fig. 3G showed a compara- tive account of the cyanocobalamin content at different developing stages of date palm fruits. We have screened water soluble vitamin content in fruits of six date palm cultivars at their developing stages (immature, semimature and mature). In present investigation, we found that vitamin production was cultivar specic and fruit develop- ing stage dependent. Vitamins B1, B3, B5, B6 were maximum in mature fruits of Shishi, Zardai, Shishi, and Muzati cultivars; however, B2, B9, B12 were found to be the highest in Khalasah, Khalasah and Shishi at the immature stage of developing fruits. The variation in vitamins yield noticed in the present investigation might be due to the change in climatic condition and the genetic pool of a particular type of the cul- tivar, which directly inuenced metabolic pathways. The occurrence of the studied vitamins with developing stages of date palm fruit agreed with the previous nding in some other plants, whereas, it was conrmed that the synthesis of end products are associated with the degree of differentiation of individual cell or tissues and environmental conditions (Al-Shahib and Marshall, 2002; Pande et al., 2002; Ma et al., 2006; Mrabet et al., 2008; Bhat et al., 2008; Junaid et al., 2009, 2010a). The results observed in the present study are very similar to the study that dealt with other active principle (Datta and Srivastava, 1997; Junaid et al., 2010c), in which, in- creased and decreased accumulations of metabolites were noted at different developmental stages of the seedlings or fruits that grew, reached a steady state when they became older. The present results also agreed with the mineral analysis carried out in various date palm cultivars, whereas, no con- stant trends were found at the developmental stages of differ- ent date palm cultivars fruit (Attalla et al., 1988; Al-Shahib and Marshall, 2002). In the present study results also showed a great variation in water soluble vitamin contents within the fruit developing stages and date palm cultivars. Similarly, in other plant system it has also been established that the meta- bolic pathway was not only restricted to certain tissues, but it was also modulated by different developmental and environ- mental conditions (Pasquali et al.,1992; Yohe, 2002; Adebiyi et al., 2005; Junaid et al., 2010b). Secondly, it has also been re- ported that the mineral content of the date fruit may be inu- enced by the level of environment and the soil characteristic as well as by the amount of fertilizers used by the trees (Al-Juburi et al., 1994; Al-Hooti et al., 1997). Recent study on water sol- uble vitamins analysis indicates that water soluble vitamin pro- duction was tissue specic and environmental condition dependent (Junaid et al., 2008). 4. Conclusion A remarkable variation in water-soluble vitamins yield was no- ticed in fruits of six date palm cultivars. Matured fruits proved to be highly economical for the production of vitamin B1, B3, B5, B6; however, the remaining others were present in the B2, B9, B12. Based on the present investigation it could be con- cluded that immature and matured date palm fruits could be used as water soluble vitamins supplements for human con- sumption in the diet of vegetarians. Acknowledgment The rst author (Dr. Junaid Aslam) is thankful to the Chair- man, Board of Trustees of Dubai Pharmacy College for pro- viding all facilities to carry out the present research work. References Adebiyi, A.O., Adebivi, A.P., Olaniyi, E.O., 2005. African J. Biotech- nol. 4, 10891094. Ahmed, I.A., Ahmed, A.W.K., Robinson, R.K., 1995. Food Chem. 54, 305309. Al-Farsi, M., Alasalvar, C., Morris, A., Baron, M., Shahidi, F., 2005. J. Agri. Food Chem. 53, 75867591. Al-Hooti, S., Sidhu, J.S., Qabazard, H., 1997. Plant Foods Hum. Nutr. 50 (2), 101113. Al-Juburi, H.J., Al-Mesry, H., Al-Banna, M., 1994. Bull. Fac. Agric. Univ. Cairo 45, 165186. Al-Shahib, W., Marshall, R.J., 2002. Int. J. Food Sci. Technol. 37, 719721. Attalla, A.M., Harraz, F.M., 1996. Arab Gulf J. Sci. Res. 14 (3), 629 639. Attalla, A.M., Haggag, M.N., Attia, M.M., Ibrahim, A.M.F., 1988. J. Agric. Res. Tanta. Univ. 14 (1), 319331. Bhat, M.A., Ahmad, S., Junaid, A., Mahmooduzzafar, Mujib, A., 2008. Chem. Pharmac. Bull. 56 (1), 1721. Quantication of water soluble vitamins in six date palm (Phoenix dactylifera L.) cultivars fruits 15 Clarkson, P.M., 1993. Nutritional Needs in Hot Environments: Applications for Military Personnel in Field Operations. National Academy Press, Washington DC: USA. Datta, A., Srivastava, P.S., 1997. Phytochemistry 46, 135137. Fayadah, J.M., Al Showiman, S.S., 1990. Chem. Soc. Paki. 12, 84103. Jacab, R.A., Sotoudeh, G., 2002. Nutr. Clin. Care 5, 6674. Junaid, A., Mohajir, M.S., Khan, S.A., Khan, A.Q., 2008. Afr. J. Biotechnol. 7 (14), 23102314. Junaid, A., Mujib, A., Sharma, M.P., 2009. Sci. Hort. 119, 325329. Junaid, A., Mujib, A., Zohra, F., Sharma, M.P., 2010a. In Vitro Cell. Dev. Biol. Plant 46, 348353. Junaid, A., Mujib, A., Sharma, M.P., 2010b. Acta Physiol. Plant. Junaid, A., Khan, S.H., Khan, S.A., 2010c. Saudi J. Chem. Soc., doi:10.1016/j.jscs.2010.12.001. Ma, R., Ritala, A., Caldentey, K.M.O., Rischer, H., 2006. Planta Med. 72, 11421148. Mrabet, A., Ferchichi, A., Chaira, N., Mohamed, B.S., 2008. Pakistan J. Biol. Sci. 11 (7), 10031008. Myhara, R.M., Karkala, J., Taylor, M.S., 1999. J. Sci. Food Agri. 79, 13451350. Pande, D., Purohit, M., Srivastava, P.S., 2002. Plant Sci. 162, 583587. Pasquali, G., Goddijin, O.J.M., De Wall, A., Verpoorte, R., Schil- peroort, R.A., Hoge, J.H.C., Memelink, J., 1992. Plant Mol. Biol. 18, 11211131. Yohe, J., 2002. Science 57, 214. Zhao, B., Tham, S.Y., Lu, J., Lai, M.H., Lee, L.K.H., Moochhala, S.M., 2004. J. Pharm. Pharmaceut. Sci. 7 (2), 200204. 16 J. Aslam et al.