CONVERSION OF KOKUM (GARCINIA INDICA) SYRUP IN POWDER FORM FROM KOKUM EXTRACT UNDER VACUUM AND ITS HPLC ANALYSIS

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1 CONVERSION OF KOKUM (GARCINIA INDICA) SYRUP IN POWDER FORM FROM KOKUM EXTRACT UNDER VACUUM AND ITS HPLC ANALYSIS * S. A. Lamture and D. S. Bhatkhande Department of Chemical Engineering, BRACT s Vishwakarma Institute of Technology, Pune, Maharashtra, India *Author for Correspondence ABSTRACT Kokum juice is very popular in India especially in hot summer. It is delicious and has a cooling effect on body. It is refreshing drink. It not only quenches your thirst, but also helps to prevent dehydration and sunstrokes due to heat. Kokum fruit possess useful anti-oxidant, chelating, anti-cancer, anti-inflammatory, anti-bacterial, anti-fungal, cardio-protective and anti-ulcer activities. The objective of this work is to convert extracted kokum syrup in powder form. This can add a new dimension in use of kokum juice as an alternative to kokum syrup, reducing volume for transportation, increasing shelf life and convenience and usefulness in medicinal applications. The aim was also to check the composition of drink prepared from powered juice and syrup. Determine the suitable conditions for preparing the powder at which the medicinal components are more preserved. The suitable condition found out is 500 mmhg. HPLC was used for comparing and analyzing the contents. Key Words: Kokum Syrup, Vacuum Distillation, Drying, Kokum Powder, Hydroxycitric Acid and Garcinol INTRODUCTION Kokum or Garcinia indica choisy is one of the 200 species in the genus Garcinia found in the Afro-Asian countries and one of the 300 species found in India. Kokum is a slender evergreen tree found in the tropical rainforests of India, particularly Maharashtra, Karnataka, Kerala, Western Ghats, Nilgiris in Tamil Nadu, South Gujarat, Goa, West Bengal and Assam. Kokum is important member of the family Clusiaceae. The flowers of kokum are unisexual, axillary or terminal and solitary. The fruits are generally spherical, dark purple when ripe, enclosing 5 to 8 large seeds. Kokum tree starts flowering during the month of November to December and fruits are harvested during April-May (Parle & Dhamija, 2013). The fruits of the tree have an agreeable flavor and a sweetish acid taste. Kokum is valued for its numerous medicinal properties. The fruit is antihelminthics, cardiotonic, and useful in piles, dysentery, tumors, pains and heart complains. Syrup from juice is driven in bilious infections (Dushyantha et al., 2010). The important compounds of Garcinia indica are hydroxycitric acid (HCA) and garcinol. Plant organic acids are hydroxycitric acid, oxalic, malic, citric, tartaric and acetic acids as given in table 1 (Parthasarathy et al., 2012). Hydroxycitric acid (HCA) is a potent anti-obesity compound and reduces lipid and sugar level in blood. HCA lowered body fat level with no loss of body protein or lean mass in test animals that had been experimentally made obese. Thus as natural product of Garcinia the HCA is working wonder in reducing weight and obesity. HCA is a potent metabolic regulator of obesity and it also lowers blood lipids such as cholesterol and triglycerides (Onakpoya et al., 2010). Table 1: Organic acid profile of Garcinia indica Compound Gm wt % Boiling point ( 0 C) Melting point ( 0 C) Hydroxycitric acid Malic acid Oxalic acid Citric acid Tartaric acid Acetic acid Garcinol, a yellow crystalline compound, which is structurally similar to curcumin established antioxidant, anti aging and anti carcinogenic agent, by virtue of containing both phenolic hydroxyl group and the β-diketone moiety. The structure of this component is shown in fig. 1. The chemical structure of garcinol, also known as camboginol, the terminal alkene contained an unsaturated isoproprenyl substituent of molecules can undergo cyclization under acidic conditions to produce iso-garcinol latter, oxidized isomer of garcinol has also been associate with anti tumorigenic, anti obesity, anti ulcer, anti bacterial, anti 16

2 viral, anti inflammatory properties. Thus, it is increasingly becoming important industrially, commercially and medicinally (Padhye et al., 2009). Figure 1: Structure of Garcinol Acidity plays an important part in the perception of fruit quality. It affects not only the sour taste of the fruit but also sweetness, by masking the test of sugars. In recent years Garcinia is more valued as medicinal crop because of presence of HCA and Garcinia. Studies on the presence of other acids like malic acid, citric acid, oxalic acid and tartaric acid will improve the importance of crop. HPLC chromatographic studies indicated that the Garcinia indica showed a great variation in number of peaks and their relative areas. The organic acid profile is shown in table 1 (Parthasarathy et al., 2012) MATERIALS AND METHODS In the laboratory scale experiments on the kokum syrup for manufacturing of kokum powder under vacuum is performed in laboratory of VIT, Pune, which can add a new dimension in use of kokum juice as an alternative to kokum syrup juice. Kokum syrup was prepared using conventional method. The kokum fruit is cut, rinds and seeds are separated. The rinds are filled with sugar and juice was collected. For preparation of kokum powder vacuum distillation process is used. The set up as shown in fig. 2 was used. The experiments are carried out at initially atmospheric and then different vacuum pressure to find appropriate vacuum pressure. For HPLC analysis of juice different HPLC grade chemicals are used. Chemicals Acetonitrile, Phosphoric acid and Orthophosphoric acid (Merck Specialties Pvt. Ltd), Methanol (Himedia Laboratories Pvt. Ltd), Acetic acid (Loba Chemie laboratory reagents & fine chemicals), water (Thermo Fisher India Pvt. Ltd). Figure 2: Experimental set up for vacuum distillation Experimental In these experiments, first 150ml (171gm) of extracted kokum syrup was taken in round bottom flask. Initial experiment is carried out without vacuum. This showed loss of coloring and flavorings compounds so vacuum distillation process is used. Different vacuum pressure such as 300, 400, 500, 600, 650, 670 mmhg is applied for 17

3 different experiments. The kokum syrup is heated for 1 to 1 hr 40min then the sample and residue is collected in sample bottles. The obtained product is thick and waxy. The kokum powder can be obtained from this solid sample by keeping it in oven for drying still it gets completely dry and then the powder is made by crushing it. The powder is shown as in Fig. 3. The results of these experiments, density and Ph are as shown in the table 2. The density of kokum syrup is gm/ml and the ph is After performing experiments on 150 ml (171 gm) kokum syrup at different vacuum like 300, 400, 500, 600, 650, 670 mmhg we obtained 115, 127, 121, 90, 109, 95.8 gm of product respectively as shown in table 2. Figure 3: Kokum Powder Table 2: Result of experiment at different vacuum pressure Vacuum pressure (mmhg) Obtained product (gm) Density (gm/ml) ph Why vacuum is used If no vacuum is used losses of colorants and flavoring compounds are possible, so vacuum is used to prevent loss of important medicinal compounds. Different vacuum are used to find the appropriate vacuum. For preparation of kokum juice we have used the dilution ratio as 1:4 by volume by using kokum and water and it is used as drink. To prepare kokum juice from kokum syrup powder the ratio of dilution was adjusted as per the amount of powder collected from initial syrup to match the same dilution. Then analysis of flavor and taste are done by visualization and drinking. Fig. 4 shows that it appears same in colour and taste is just similar to kokum juice which is made from the extracted syrup. a) Before b) After Figure 4: Kokum juice before and after experiment 18

4 Voltage Voltage Ind. J. Sci. Res. and Tech (4):16-20/Lamture & Bhatkhande HPLC Analysis HPLC is used for analyzing different samples which are obtained before and after performing experiments. Instrumentation and chromatographic conditions The HPLC system consisted of LC model C18 column, the injection system used contained a 25 µl sample loop and UV detector. For analysis of garcinol and hydoxycitric acid, mixture of acetonitrile-water (90:10, v/v) and methanol- acetic acid (99.5:0.5, v/v) is used as mobile phase, the UV detector used is 220nm and flow rate is 0.5 ml/min. For analysis of isogarcinol, 0.1% orthophosphoric acid in water and acetonitrile (20:80, v/v) is used as mobile phase. UV detector used is as 215 nm and flow rate is 1 ml/min. The injection volume was 25 µl. the analysis of residue is also done to check the losses of the compounds. The retention time for all compounds is between 10 to 15 minutes. The retention time of HCA and garcinol are 4.31 and respectively. Sample Preparation For HPLC analysis kokum powder is considered equivalent to kokum syrup as per amount of product collected from experiment and initial amount and then diluted in same manner as juice for further analysis. The kokum juice is further diluted as 200mg/liter of total juice for detection of HCA and garcinol and 400 mg/liter of total juice for detection of isogarcinol, and then it is filtered by using filter paper. The filtration is done twice. The analysis is done for the important components which are present in the product. The distillate water is collected and also analyzed to check any loss of important compounds. RESULTS AND DISCUSSION The kokum syrup is converted in solid powder form. We observed that the suitable condition for making powder is at 500 mmhg; here the volume of the product is reduced by %. All the medicinal compounds are retained in the powder. The prepared sample analysis of the juice produced from the powder, original juice diluted and analysis of distillate water shows that the vaccum of 500 mm Hg is the best. The solid powder and residue are obtained from 150 ml (171gm) as shown in the table 3. Table 3: Result of experiment at different vacuum Vacuum pressure (mmhg) Obtained product (gm) Residue (ml) [mv] 20 c:\documents and settings\admin\desktop\kokum Time Figure 5: Chromatogram at 500 mmhg by using acetonitrile and water (30%) and methanol and acetic acid (70%) [min.] [mv] C:\Documents and Settings\admin\Desktop\kokum garcinol Figure 6: Chromatogram at 500 mmhg by using 0.1% orthophosphoric acid Time [min.] 19

5 The HPLC analysis is done to check whether the compounds of kokum juice remains similar which is obtained after the experimental juice. The main ingredients of kokum are garcinol, isogarcinol and Hydroxycitric acid. It also contains oxalic acid, tartaric acid, malic acid and citric acid. The detection HCA and Garcinol were confirmed by determination of relative retention time as shown in fig. 5. The retention time is 4.31 and respectively. Also the detection of isogarcinol, peak was confirmed. The retention time of isogarcinol is in between 6.8 to 7.01 min as shown in fig. 6. The run time of HPLC was kept for 10 to 15 min. small peaks of oxalic acid, tartaric acid, malic acid and citric acid are detected in the time 2.6 to 2.9, 3 To 3.5, 3.9 to 4.2, and 7.2 to 7.58 respectively that shows their presence in the kokum powder. CONCLUSION The extracted kokum syrup can be easily converted in powder form under vacuum, 500 mmhg is best vacuum because all medicinal compounds are retained in the powder and very less losses of colorant and flavoring components are observed by using vacuum distillation, so that it can add a new dimension in use of kokum juice as an alternative to kokum syrup juice. The compounds, which is present in kokum powder juice remains same like kokum juice which is made from syrup and it is confirmed by using HPLC method. The volume of product is reduced 35.67%. The importance of solid powder is as it can be easily packed in small pouches and easy to transportation. The kokum powder also can be used in weight reducing capsules. Garcinia is sold in many different forms, from capsules and tablets to powder that can mix with water or juice for refreshing drink, so Garcinia indica juice is healthier and far more refreshing option as compared to commercial bottled drinks. Hence generation of powder can be new business opportunity for Garcinia indica growing farmers in India. REFERENCES Braganzo M, Shirodkar A, Jayarama Bhat D & Krishnan S (2012). Western Ghats Resource Book on kokum. Panaji-Goa, Western Ghats kokum Foundation. Daniel JN & Dudhade PA (2006). Analysis of economic characteristics of volume chain of three underutilized fruits of India. Research Report No.3, International center for Underutilized crops, Colombo, Sri Lanka 4-8. Dushyantha D, Girish D & Suvarna V (2010). Native lactic acid bacterial isolates of kokum for preparation of fermented beverage. EJournal of Biological Sciences, 2(1) 1-4. Jayaprakasha G, Jena B, & Sakariah K (2003). Improved Liquid Chromatographic Method for Determination of Organic Acid in Leaves, Pump, Fruits, and Rinds of Garcinia. Journal of AOAC International, 86(5) 1-6. Kumar S, Sharma S & Chattopadhyay S (2013). Rapid and sensitive HPLC-PDA method for simultaneous identification and qualification of dietary weight reducing compound Hydroxycitric acid lactone and chemo preventive compounds isoxanthochymol and xanthochymol in garcinia indica. International Food Journal, 20(1) Kureel RS, Kishor R, Pandey A & Dutta A (2009). Kokum a potential tree borne oilseed, national oilseeds and vegetative oils development board, ministry of agriculture Gov. of India Lemos NM (2011). Evaluation of neuroprotective and analgesic role of garcinia indica choisy aqueous fruit extract in different experimental animal models. Collage of Pharmacy Belgaum, Karnataka, India Onakpoya I, Hung S, Perry R, Wider B & Ernst E (2010). The Use of Garcinia extract (hydoxycitric acid) as a weight loss supplement: A Systematic review and meta-analysis of randomized clinical trials. Journal of Obesity, 1-5. Padhey S, Ahmad A, Oswal N & Sarkar F (2009). Emerging role of Garcinol, the antioxidant chalcone from Garcinia indica Choisy and its synthetic analogs. Journal of Hematology & Oncology, 2(38) Parle M & Dhamija I (2013). Golden benefits of drinking kokum-cola. International Research Journal of Pharmacy, 4(5) 5-9. Parthasarathy U, Nandakishore O, Senthil Kumar R, Nirmal Babu K, Zachariah T & Paethasarathy V (2012). Chromatographic fingerprinting and estimation of organic acids in selected species. International Journal of Innovative Horticulture, 1(1) Praveen Kumar SN, Bhadre Gowda DG, Mantelingu K & Rangappa KS (2013). Development and Validation of a Reversed-Phase HPLC method for Analysis of Garcinol and isogarcinol in Garcinia indica. Journal of Pharmacy Research, Saadat N & Gupta S (2012). Potential Role of Garcinol as an Anticancer Agent. Journal of Oncology,