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Extraction of forskolin from Coleus forskohlii roots using three phase partitioning
Separation and Purification Technology 96 (2012) 20–25
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Extraction of forskolin from Coleus forskohlii roots using three phase partitioning Shirish M. Harde, Rekha S. Singhal ⇑ Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India
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Article history: Received 25 February 2012 Received in revised form 3 May 2012 Accepted 10 May 2012 Available online 18 May 2012 Keywords: Coleus forskohlii root Forskolin Extraction Ultrasonication Enzyme assisted three phase partitioning
a b s t r a c t Three phase partitioning (TPP), a technique which is based on partitioning of hydrophilic constituents, proteins, and hydrophobic constituents in three phases comprising of water, ammonium sulphate and organic solvent, was explored for extraction of diterpene, forskolin from Coleus forskohlii roots. The process which consists of simultaneous addition of t-butanol and ammonium sulphate to the aqueous slurry of C. forskohlii roots was optimized with respect to the concentration of ammonium sulphate loading and the ratio of t-butanol to slurry. A maximum of 30.83% recovery of forskolin was obtained under the optimized conditions. Ultrasonication and enzyme pretreatment with commercial enzyme preparation of StargenÒ 002 and AccelleraseÒ 1500 followed by TPP gave 79.95% and 83.85% recovery when used individually within 4 h as compared to 12 h in Soxhlet extraction. A combination of the two pretreatments increased the yield marginally. Hence enzymatic pretreatment followed by TPP is recommended for extraction of forskolin. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Forskolin (FSK), a labdane diterpene compound isolated from the roots of Coleus forskohlii Briq. [1], is useful in the treatment of health disorders including cardiovascular diseases, hypertension [2,3], asthma, glaucoma [4] and Alzheimer’s disease [5]. Its further use in promoting lean body mass, treating mood disorders and its anticancer activities [6] is well known. The pharmacological activities of FSK are mainly due to its role as an activator of adenylate cyclase. FSK increases the amount of cyclic AMP (cAMP) (adenosine monophosphate) in cells by activating adenylate cyclase enzyme. cAMP is an important secondary messengers in the cell, and is considered to be an effective cell regulating compound [7]. The structural complexity of FSK makes the chemical synthesis difficult and uneconomical. Besides, synthetic forskolin is reportedly not as effective as that procured from the natural source [8]. Among the several methods described in the literature for extraction, isolation and purification of FSK from the roots of C. forskohlii, refluxing the powdered roots in multiple extraction steps with organic solvents such as benzene, methanol, chloroform and toluene [9] is commonly used commercially. This method gives an extract that is sticky and viscous in nature, besides being time consuming and requiring relatively large quantities of solvents [10]. Supercritical fluid extraction is a potential alternative to conventional extraction methods using organic solvents for extracting biologically active components from plants [11]. However, this method requires expensive high pressure equipment and may also require ⇑ Corresponding author. Tel.: +91 22 3361 2512; fax: +91 22 3361 1020. E-mail address: [email protected] (R.S. Singhal). 1383-5866/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.seppur.2012.05.017
organic solvents as co-solvent for complete extraction of the bioactives [12]. Other methods reported for extraction of forskolin are hydrotropic extraction [12] and microwave assisted extraction [13], of which the former does not give sufficiently pure FSK. In this paper, we report an alternative extraction process for the recovery of FSK using three phase partitioning (TPP). TPP is a relatively recent bioseparation technique in which a suitable amount of ammonium sulphate and t-butanol is added to an aqueous suspension of the sample. The protein precipitates out in the middle layer between the organic and aqueous phases due to multiple phenomena such as salting out, isotonic precipitation, co-solvent precipitation, osmolyte precipitation and kosmotropic precipitation. Polar compounds such as saccharides separate out in the lower aqueous layer, while non-polar compounds such as oils separate in the upper organic layer. TPP is a simple, inexpensive, scalable, and rapid procedure, works at room temperature, and the chemicals used in the process can be recycled. It does not use polymers which have to be removed later. Tertiary butanol is normally completely miscible with water (b.p. 84 °C) and much less flammable than hexane, methanol or ethanol which are used in conventional extraction. All developments on TPP so far have used t-butanol. TPP has been used for both upstream and downstream processing of enzymes such as bifunctional protease/amylase inhibitor [14], invertase [15], a-galactosidase [16,17], and serine protease [18]. However, literature on use of TPP for extraction of oleaginous material is scant. Sharma et al. [19] showed TPP to extract 82% oil from soybean oil within 1 h, while Shah et al. [20] reported it to extract 97% oil from Jatropha curcas L. within 2 h. We reported on successful application of TPP in extraction of oleoresin from turmeric [21].
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Pretreatments using enzymes, either alone [22,23] or in combination with sonication [20] has shown increased yields of oil from soybean, rice bran, mango kernel, peanuts and jatropa. Similarly, ultrasonication followed by TPP (UATTP) has shown 87%, 77% and 88% recovery of oil from almond, apricot and rice bran, respectively [24]. Enzyme pretreatment of plant material could help in breakdown of cellulosic structure to increase the extraction efficiency of FSK. Shah et al. [20] used enzyme pretreatment prior to TPP to increase the yield of oil and called this approach as enzyme-assisted three phase partitioning (EATPP). The efficiency of EATPP was concluded to be comparable to solvent extraction with added advantage of being less time consuming. Since FSK is soluble in polar solvents [25], we hypothesized that it may be possible to extract it from C. forskohlii roots by TPP. The present work demonstrates the application of TPP in extraction of FSK from C. forskohlii roots. An important further step in the process development has been the pretreatment of the plant material with ultrasonication and enzymes prior to TPP. 2. Materials and methods 2.1. Materials C. forskohlii roots were procured from Salem, Tamilnadu, India. Dried roots were ground in a mill fitted with 18 mesh sieve to get a particle size below 1 mm and stored in an air tight container for further studies. Standard forskolin (FSK) was a gift sample from Medicinal and Natural Product Research Laboratory, Institute of Chemical Technology, Mumbai, India. Methanol, t-butanol and ammonium sulphate were procured from S. D. Fine Chemicals Limited, Mumbai, India. Enzyme samples (StargenÒ 002 and AccelleraseÒ 1500) were gifted by Genencor International, Mumbai, India. 2.2. Three phase partitioning (TPP) TPP was optimized by varying the ammonium sulphate loading and ratio of t-butanol to slurry i.e. the quantity of solvent required for maximum yield of extractives. Slurry was prepared by dispersing 5 g C. forskohlii root powder in 50 mL distilled water by gentle stirring using a magnetic stirrer. Weighted amount of ammonium sulphate was added to the slurry prepared and vortexed gently, followed by addition of measured amount of t-butanol. The extraction was carried out for 1 h by gentle stirring with magnetic stirrer. The mixture was allowed to stand for 1 h for the formation of three phases. The three phases so formed were separated by centrifugation at 5000 g for 20 min. The upper organic layer was collected and the solvent (t-butanol) was evaporated on a rotary vacuum evaporator under reduced pressure at 50 °C for 2 min (Buchi Rotavapor, R-124, Switzerland). The extract so obtained was quantified for FSK content by using HPLC. Ammonium sulphate loading was varied from 10 to 50% w/v of slurry while ratio of t-butanol to slurry was varied from 0.5:1 to 2:1 with all the other extraction conditions being unchanged. Further evaluation of extraction time from 30 to 120 min was carried out for maximum yield of FSK. To check the effect of pH on extraction of FSK, pH of the system was adjusted to pH 4, 5, 6 and 7 by the addition of 1 N HCl and 1 N NaOH after addition of ammonium sulphate to the slurry. The concentrations of ammonium sulphate and t-butanol thus optimized were used further for EATPP and UATPP. 2.3. Enzyme assisted three phase partitioning (EATPP) One factor at-a-time method was used to study the effect of various parameters on the extraction of FSK. Temperature and pH
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were kept constant at their optimal levels as determined experimentally (data not shown). The parameters were studied individually for StargenÒ 002 (pH 4.5, 50 °C and 2000 U/mL) and AccelleraseÒ 1500 (pH 4.5, 50 °C and 1500 FPU/mL). One unit of enzyme activity was defined as the amount of enzyme that would liberate one mole of reducing sugar per minute under optimized assay conditions. StargenÒ 002 is a blend of a-amylase and glucoamylase which hydrolyses starch. AccelleraseÒ 1500 is a mixture of cellulase and glucosidase having action on cellulose. In EATPP, enzyme pretreatment was given to C. forskohlii root slurry prior to TPP using StargenÒ 002 and AccelleraseÒ 1500 in Mcllvaine’s buffer. Specific amount of enzyme was added to the slurry, and the slurry was incubated with gentle stirring with a magnetic stirrer for 1 h. After incubation, FSK was extracted by TPP using the optimized conditions as determined earlier, and the extract was quantified for FSK. For StargenÒ 002 and AccelleraseÒ 1500 pretreatment, enzyme concentration (16–80 U/g of substrate and 30–150 FPU/g of substrate, respectively) were optimized. Effect of combination of these two enzymes was also studied. For this, slurries were incubated for 1 h after addition of StargenÒ 002 and AccelleraseÒ 1500 (64 U/g of substrate and 90 FPU/g of substrate, respectively) at pH 4.5, other conditions of extraction by EATPP being the same. Slurries were prepared in Mcllvaine’s buffer system [26]. Incubation time of combined StargenÒ 002 and AccelleraseÒ 1500 pretreatment was optimized by incubating the slurries (pH-4.5, 50 °C) for 30–120 min. The extract so obtained was quantified for FSK content. 2.4. Ultrasound assisted three phase partitioning (UATPP) In UATPP, ultrasound pretreatment was given to C. forskohlii root slurry prior to TPP. Optimization was carried out by using one factor at-a-time method. Optimization of power requirement for the maximum extraction of FSK after TPP was done by varying the power from 18 to 58 W for 10 min. Effect of duty cycle (30–70%) as a pretreatment on extraction of FSK evaluated by subjecting 10% w/v of slurry prepared in distilled water at optimized power for 10 min. Further, optimization of extraction time (5–20 min) of sonication before TPP was carried out for maximum extraction of FSK. The extract was then quantified for FSK content. 2.5. Combination of ultrasonication and enzyme pretreatment followed by TPP To check the combined effect of ultrasonication and enzyme pretreatment, C. forskohlii root slurry was treated with optimized parameters obtained from ultrasonication study. Further, ultrasound treated slurry was subjected to enzyme pretreatment followed by TPP. The extract was then quantified for FSK content. 2.6. Conventional solvent extraction The C. forskohlii root powder was extracted in a Soxhlet apparatus using methanol for 12 h. The extract was cooled and then concentrated by evaporating in rotary vaccum evaporator under reduced pressure at 50 °C (Buchi Rotavapor, R-124, Switzerland). Solvent was recovered from rotavac. The FSK yield was expressed as % w/w of C. forskohlii root powder. 2.7. Analytical determination A Jasco HPLC system fitted with Zorbax eclipse XDB C18 column (5l 4.6 mm 250 mm) was used. The column was equilibrated with an acetonitrile-water (50:50) mixture as mobile phase at a flow rate of 1.5 mL/min. FSK was detected by measuring UV absorption at 217 nm [27]. Retention time of standard FSK was 9.7 min.
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The basic phenomenon of TPP is that the water/t-butanol mixture separates a crude proteinaceous extract into a protein-rich middle layer in addition to the two liquid phases. An increased activity of enzymes is often seen in this middle layer [28]. Pigments, lipids and enzyme inhibitors are concentrated in upper phase, while polar components like saccharides are enriched in the lower aqueous phase. Solvent extraction of C. forskohlii roots gave a FSK yield of 0.64 ± 0.12% w/w of substrate which was considered as 100% recovery and against which the FSK recovery by TPP was estimated. Fig. 1a–c represents the effect of ammonium sulphate loading, quantity of t-butanol and extraction time on the yield of FSK expressed as % w/w of C. forskohlii root powder, respectively. The maximum FSK yield of 0.194 ± 0.0025% w/w (corresponding to extraction efficiency of 30.31%) was achieved with 30% w/v of ammonium sulphate. Many other researchers reported 30% w/v ammonium sulphate to give best results where TPP has been used for separation of proteins/enzymes [29]. Sharma et al. [19] also found that 30% w/v of ammonium sulphate to show optimum extraction of soybean oil by TPP. Fig. 1b shows the effect of varying the ratio of t-butanol and aqueous slurry of C. forskohlii root. Decreasing the t-butanol volume by half decreased the FSK yield whereas increasing t-butanol to twice the volume did not lead to any significant increase. Thus, 1:1 ratio was employed in further studies. Optimum extraction time was found to be 60 min. FSK yield obtained was 0.197 ± 0.003% w/w with extraction efficiency of 30.83% under the optimum conditions of TPP. We prepared the slurry of root powder in distilled water (pH-7.0) and pH of the system was found to be 5.2. After addition of ammonium sulphate to the slurry, pH of the system increased to 5.5. The influence of pH on extraction yield was studied by changing the pH of the system from 4 to 7 after addition of ammonium sulphate to the slurry. The yield of FSK at different pH values (Fig. 1d) did not change significantly. Further experiments were performed under these optimum conditions of TPP without adjusting the pH of the system. 3.2. Enzyme assisted three phase partitioning (EATPP) FSK is a terpenoid which is mainly situated in cytoplasmic vesicles of roots. The root is mainly divided into outermost cork cells followed by cortex, cambium, and xylem. Cork consists of polygonal cells. Yellowish to brown masses are found in the cells of cork, cortex, medullary rays, and xylem. These are identified as cytoplasmic vesicles containing secondary metabolites/terpenoids [30]. These cytoplasmic vesicles are generally 5–12 lm in diameter and are attached to the outer wall of the cork cells by a membranous stalk. The location of FSK in such as cell structure is likely to offer some resistance to penetration by solvent before enabling its solubilization in solvents. At higher temperatures, an increased rupture of the cellular structure is possible by hydrolysis of cellulose following increased solubilization of solute into the solution which may reduce the firmness of the cell wall. It is well established that terpenoids are trapped in cytoplasmic vesicles. The cytoplasmic vesicles are present in both the bark and wood regions.
Forskolin (% w/w of substrate)
3.1. Three phase partitioning (TPP)
0.2 0.15 0.1 0.05 0
10
20
30
40
50
Ammonium sulphate loading (% w/v of slurry)
(b) Foskolin (% w/w of substrate)
3. Results and discussion
(a) 0.25
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1
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Ratio of t -butanol to slurry (% v/v of slurry)
(c) Forskolin (% w/w of substrate)
Quantification of FSK was done by extrapolating the area under the curve to the concentration of standard FSK.
0.25 0.2 0.15 0.1 0.05 0
30
60
90
120
6
7
Time (min)
(d) Forskolin (% w/w of substrate)
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0.22 0.2 0.18 0.16 0.14 0.12 0.1
4
5
pH Fig. 1. Optimization of parameters for three phase partitioning (a) effect of ammonium sulphate loading with t-butanol to slurry (1:1) and extraction time of 60 min, (b) ratio of t-butanol to slurry with 30% ammonium sulphate loading and extraction time of 60 min, (c) effect of extraction time with 30% ammonium sulphate loading and t-butanol to slurry (1:1), and (d) effect of pH with 30% ammonium sulphate loading, t-butanol to slurry (1:1) and extraction time of 60 min.
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Quantification of FSK in different tissues has shown terpenoids to be mainly concentrated in the woody layer [31]. Thus, it was considered worthwhile to attempt pretreatment of the C. forskohlii
(a) 0.3 0.25 0.2 0.15 0.1 0.05 0
control
16 32 48 64 Stargen conc. (U/gm of substrate)
80
Forskolin (% w/w of substrate)
(b) 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0
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30 60 90 120 Accellerase conc. (FPU/gm of substrate)
150
F0rskolin (% w/w of substrate)
Forskolin (% w/w of substrate)
0.35
root slurry by enzymes before carrying out TPP. Aqueous extraction of oils using enzymes has been successfully used in extraction of oils from rice bran [32] and peanut [23]. EATPP has not been attempted for extraction of FSK from C. forskohlii roots so far. StargenÒ 002 enzyme contains Aspergillus kawachi alpha-amylase expressed in Trichoderma reesei and a glucoamylase from T. reesei that work synergistically to hydrolyze granular starch substrate to glucose. AccelleraseÒ 1500 is a mixture of cellulase and glucosidase that works synergistically to hydrolyze cellulosic substrate to glucose. As C. forskohlii root contained 14– 15% cellulose and 18–20% starch (determined earlier in the laboratory), these two enzymes could increase the efficiency of TPP by breaking complex cellulosic matrix that entraps FSK and thereby increase the release of FSK. Extraction of FSK carried out by varying the concentration of StargenÒ 002 showed optimum extraction of FSK at 64 U/g of substrate (Fig. 2a). Similarly, AccelleraseÒ 1500 showed maximum extraction efficiency at 90 FPU/g of substrate (Fig. 2b). With further increase in concentration, no increase in extraction efficiency was observed. Pretreatments with StargenÒ 002 and AccelleraseÒ 1500
0.35 0.3 0.25 0.2 0.15 0.1 0.05 0
control
Forskolin (% w/w of substrate)
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Stargen (S)
Accellerase (A)
S+A
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50
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120
Time (min) Fig. 2. Optimization of parameters for enzyme assisted three phase partitioning (a) effect of StargenÒ 002 concentration with incubation time of 60 min, (b) effect of AccelleraseÒ 1500 concentration with incubation time of 60 min, (c) combined effect of enzymes with incubation time of 60 min, and (d) Effect of incubation time of combined StargenÒ 002 and AccelleraseÒ 1500.
Forskolin (% w/w of substrate)
Forskolin (% w/w of substrate)
36
(b) 0.5
0.5
(d) Forskolin (% w/w of substrate)
28
Power output (Watts)
(c) 0.6
0
18
0.6 0.5 0.4 0.3 0.2 0.1 0
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5
10
15
20
Time (min) Fig. 3. Optimization of parameters for ultrasound assisted three phase partitioning (a) effect of Power output, (b) effect of duty cycle, and (c) effect of extraction time.
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Table 1 Extraction of forskolin: comparison between conventional solvent extraction, UATPP, EATPP and UATPP + EATPP.
a
Parameter
Conventional solvent extractiona
UATPPa
EATPPa StargenÒ 002 + AccelleraseÒ 1500
UATPP + EATPPa
Solvent Extraction time (h) Yield of FSK (% w/w of Coleus forskohlii roots) Extraction efficiency (%)
Methanol 12 0.64 ± 0.12 –
t-Butanol 4 0.51 ± 0.01 79.95 ± 1.63
t-Butanol 4 0.53 ± 0.01 83.85 ± 2.39
t-Butanol 4 0.55 ± 0.004 85.16 ± 0.78
Results are mean ± standard deviation of three determinations.
individually gave maximum FSK yield of 0.27 ± 0.004% w/w and 0.45 ± 0.01% w/w, corresponding to extraction efficiency of 42.71 and 70.31%, respectively. Combined effect of StargenÒ 002 and AccelleraseÒ 1500 improved the yield to 0.53 ± 0.01% w/w with extraction efficiency of 83.85% (Fig. 2c). Evaluation of incubation time from 30 to 120 min showed 60 min to be sufficient for maximum extraction of FSK (Fig. 2d). Sharma et al. [32] reported that combination of protease, cellulase and amylase as a pretreatment yielded 77% recovery of rice bran oil. Extraction of peanut oil with protizyme pretreatment could increase the recovery upto 92% [23]. Shah et al. [20] reported that extraction of oil from Jatropa curcas L. with fungal proteases could increase the recovery upto 97%.
4. Conclusion Enzyme pretreatment followed by three phase partitioning is a promising approach of obtaining efficient extraction of FSK from C. forskohlii roots. Ultrasonication as a pretreatment did increase the extraction of FSK when used individually but was not very effective in further increasing the extraction of FSK when used in combination with enzyme assisted three phase partitioning. Hence, enzyme assisted three phase partitioning can be recommended for extraction of FSK. Unlike the conventional solvent extraction, which requires 12 h for complete extraction, the method developed herein takes only about 4 h for comparable yields of extractives. Acknowledgement
3.3. Ultrasound assisted three phase partitioning (UATPP) Ultrasonication has been used as a pretreatment for extraction of nutraceuticals from plants having advantages like reduced extraction time, reduced organic solvent consumption, and minimizing pollution [33,34]. Ultrasonication efficiently disintegrates bigger particles into smaller ones [35]. Hence, we first optimized this process for maximum extraction of FSK in terms of operational parameters and followed it with TPP as optimized in Section 3.1 as above. Fig. 3a–c showed a power output of 50 W, a duty cycle of 50%, and extraction time of 15 min to give maximum FSK yields of 0.368 ± 0.002% w/w, 0.46 ± 0.007% w/w, and 0.51 ± 0.01% w/w, corresponding to extraction efficiencies of 57.50%, 72.13% and 79.95%, respectively. These results are in the range of 77–88% recovery of oils from almond, apricot and rice bran, respectively, using a similar approach [24].
3.4. Combination of ultrasonication and enzyme pretreatment followed by TPP Ultrasonication with enzyme pretreatment increased the FSK yield to 0.55 ± 0.004% w/w with extraction efficiency of 85.16% (Table 1). Shah et al. [20] reported a combination of sonication and enzyme treatment with a commercial preparation of fungal proteases at pH 9.0 to give a yield of 97% oil within 2 h. A similar approach of ultrasonication followed by EATPP was followed by Gaur et al. [22] for extraction of edible oils from mango kernel, soybean and rice bran. The total time required for extraction of FSK using the combined pretreatment followed by TPP was about 4 h while conventional solvent extraction needed 12 h for similar comparable yield. FSK in the upper organic phase and middle layer was found to be 0.55 ± 0.004% w/w and 0.08 ± 0.004% w/w, respectively, while it was absent in the lower aqueous phase. Table 1 summarizes the yields of FSK obtained with TPP, EATPP, UATTP and UATTP + EATTP under the optimized conditions. It is evident that ultrasonication prior to EATPP increased the percent recovery of FSK only marginally which may not justify including this step in the extraction protocol. Hence, EATTP can be concluded to be a feasible and rapid technique for extraction of FSK. However, extensive scale-up studies are required for its use at industrial level.
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[27] S. Mukherjee, B. Ghosh, S. Jha, Establishment of forskolin yielding transformed cell suspension cultures of Coleus forskohlii as controlled by different factors, J. Biotechnol. (2000) 73–81. [28] E. Kiss, J. Szamos, B. Tamas, R. Borbas, Interfacial behavior of proteins in three phase partitioning using salt containing water/tert-butanol systems, Colloids Surf. 142 (1998) 295–302. [29] A. Sharma, M.N. Gupta, Purification of pectinases by three phase partitioning, Biotechnol. Lett. 23 (2001) 1625–1627. [30] R. Wongkittipong, L. Pratt, S. Damronglerd, C. Gourdon, Solid-liquid extraction of andrographolide from plants-experimental study, kinetic reaction, and model, Sep. Purif. Technol. 40 (2004) 147. [31] P. Narayanan, K. Laddha, K. Akamanchi, Histochemical localization of forskolin and other terpenoids in Coleus forskohlii, Curr. Sci. 83 (2002) 945–946. [32] A. Sharma, S.K. Khare, M.N. Gupta, Enzyme assisted aqueous extraction of rice bran oil, J. Am. Oil Chem. Soc. 78 (2001) 949–951. [33] M. Vinatoru, An overview of the ultrasonically assisted extraction of bioactive principles from herbs, Ultrason. Sonochem. 8 (2001) 303–313. [34] T.J. Mason, L. Paniwnyk, J.P. Lorimer, The uses of ultrasound in food technology, Ultrason. Sonochem. 3 (1996) 253–260. [35] K. Szentmihalyi, P. Vinkler, B. Lakatos, I. Vendel, M. Then, Rose hip (Rosa canina L.) oil obtained from waste hip seeds by different extraction methods, Bioresour. Technol. 82 (2002) 195–201.
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Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Extraction of forskolin from Coleus forskohlii roots using three phase partitioning Shirish M. Harde, Rekha S. Singhal ⇑ Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India
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Article history: Received 25 February 2012 Received in revised form 3 May 2012 Accepted 10 May 2012 Available online 18 May 2012 Keywords: Coleus forskohlii root Forskolin Extraction Ultrasonication Enzyme assisted three phase partitioning
a b s t r a c t Three phase partitioning (TPP), a technique which is based on partitioning of hydrophilic constituents, proteins, and hydrophobic constituents in three phases comprising of water, ammonium sulphate and organic solvent, was explored for extraction of diterpene, forskolin from Coleus forskohlii roots. The process which consists of simultaneous addition of t-butanol and ammonium sulphate to the aqueous slurry of C. forskohlii roots was optimized with respect to the concentration of ammonium sulphate loading and the ratio of t-butanol to slurry. A maximum of 30.83% recovery of forskolin was obtained under the optimized conditions. Ultrasonication and enzyme pretreatment with commercial enzyme preparation of StargenÒ 002 and AccelleraseÒ 1500 followed by TPP gave 79.95% and 83.85% recovery when used individually within 4 h as compared to 12 h in Soxhlet extraction. A combination of the two pretreatments increased the yield marginally. Hence enzymatic pretreatment followed by TPP is recommended for extraction of forskolin. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Forskolin (FSK), a labdane diterpene compound isolated from the roots of Coleus forskohlii Briq. [1], is useful in the treatment of health disorders including cardiovascular diseases, hypertension [2,3], asthma, glaucoma [4] and Alzheimer’s disease [5]. Its further use in promoting lean body mass, treating mood disorders and its anticancer activities [6] is well known. The pharmacological activities of FSK are mainly due to its role as an activator of adenylate cyclase. FSK increases the amount of cyclic AMP (cAMP) (adenosine monophosphate) in cells by activating adenylate cyclase enzyme. cAMP is an important secondary messengers in the cell, and is considered to be an effective cell regulating compound [7]. The structural complexity of FSK makes the chemical synthesis difficult and uneconomical. Besides, synthetic forskolin is reportedly not as effective as that procured from the natural source [8]. Among the several methods described in the literature for extraction, isolation and purification of FSK from the roots of C. forskohlii, refluxing the powdered roots in multiple extraction steps with organic solvents such as benzene, methanol, chloroform and toluene [9] is commonly used commercially. This method gives an extract that is sticky and viscous in nature, besides being time consuming and requiring relatively large quantities of solvents [10]. Supercritical fluid extraction is a potential alternative to conventional extraction methods using organic solvents for extracting biologically active components from plants [11]. However, this method requires expensive high pressure equipment and may also require ⇑ Corresponding author. Tel.: +91 22 3361 2512; fax: +91 22 3361 1020. E-mail address: [email protected] (R.S. Singhal). 1383-5866/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.seppur.2012.05.017
organic solvents as co-solvent for complete extraction of the bioactives [12]. Other methods reported for extraction of forskolin are hydrotropic extraction [12] and microwave assisted extraction [13], of which the former does not give sufficiently pure FSK. In this paper, we report an alternative extraction process for the recovery of FSK using three phase partitioning (TPP). TPP is a relatively recent bioseparation technique in which a suitable amount of ammonium sulphate and t-butanol is added to an aqueous suspension of the sample. The protein precipitates out in the middle layer between the organic and aqueous phases due to multiple phenomena such as salting out, isotonic precipitation, co-solvent precipitation, osmolyte precipitation and kosmotropic precipitation. Polar compounds such as saccharides separate out in the lower aqueous layer, while non-polar compounds such as oils separate in the upper organic layer. TPP is a simple, inexpensive, scalable, and rapid procedure, works at room temperature, and the chemicals used in the process can be recycled. It does not use polymers which have to be removed later. Tertiary butanol is normally completely miscible with water (b.p. 84 °C) and much less flammable than hexane, methanol or ethanol which are used in conventional extraction. All developments on TPP so far have used t-butanol. TPP has been used for both upstream and downstream processing of enzymes such as bifunctional protease/amylase inhibitor [14], invertase [15], a-galactosidase [16,17], and serine protease [18]. However, literature on use of TPP for extraction of oleaginous material is scant. Sharma et al. [19] showed TPP to extract 82% oil from soybean oil within 1 h, while Shah et al. [20] reported it to extract 97% oil from Jatropha curcas L. within 2 h. We reported on successful application of TPP in extraction of oleoresin from turmeric [21].
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Pretreatments using enzymes, either alone [22,23] or in combination with sonication [20] has shown increased yields of oil from soybean, rice bran, mango kernel, peanuts and jatropa. Similarly, ultrasonication followed by TPP (UATTP) has shown 87%, 77% and 88% recovery of oil from almond, apricot and rice bran, respectively [24]. Enzyme pretreatment of plant material could help in breakdown of cellulosic structure to increase the extraction efficiency of FSK. Shah et al. [20] used enzyme pretreatment prior to TPP to increase the yield of oil and called this approach as enzyme-assisted three phase partitioning (EATPP). The efficiency of EATPP was concluded to be comparable to solvent extraction with added advantage of being less time consuming. Since FSK is soluble in polar solvents [25], we hypothesized that it may be possible to extract it from C. forskohlii roots by TPP. The present work demonstrates the application of TPP in extraction of FSK from C. forskohlii roots. An important further step in the process development has been the pretreatment of the plant material with ultrasonication and enzymes prior to TPP. 2. Materials and methods 2.1. Materials C. forskohlii roots were procured from Salem, Tamilnadu, India. Dried roots were ground in a mill fitted with 18 mesh sieve to get a particle size below 1 mm and stored in an air tight container for further studies. Standard forskolin (FSK) was a gift sample from Medicinal and Natural Product Research Laboratory, Institute of Chemical Technology, Mumbai, India. Methanol, t-butanol and ammonium sulphate were procured from S. D. Fine Chemicals Limited, Mumbai, India. Enzyme samples (StargenÒ 002 and AccelleraseÒ 1500) were gifted by Genencor International, Mumbai, India. 2.2. Three phase partitioning (TPP) TPP was optimized by varying the ammonium sulphate loading and ratio of t-butanol to slurry i.e. the quantity of solvent required for maximum yield of extractives. Slurry was prepared by dispersing 5 g C. forskohlii root powder in 50 mL distilled water by gentle stirring using a magnetic stirrer. Weighted amount of ammonium sulphate was added to the slurry prepared and vortexed gently, followed by addition of measured amount of t-butanol. The extraction was carried out for 1 h by gentle stirring with magnetic stirrer. The mixture was allowed to stand for 1 h for the formation of three phases. The three phases so formed were separated by centrifugation at 5000 g for 20 min. The upper organic layer was collected and the solvent (t-butanol) was evaporated on a rotary vacuum evaporator under reduced pressure at 50 °C for 2 min (Buchi Rotavapor, R-124, Switzerland). The extract so obtained was quantified for FSK content by using HPLC. Ammonium sulphate loading was varied from 10 to 50% w/v of slurry while ratio of t-butanol to slurry was varied from 0.5:1 to 2:1 with all the other extraction conditions being unchanged. Further evaluation of extraction time from 30 to 120 min was carried out for maximum yield of FSK. To check the effect of pH on extraction of FSK, pH of the system was adjusted to pH 4, 5, 6 and 7 by the addition of 1 N HCl and 1 N NaOH after addition of ammonium sulphate to the slurry. The concentrations of ammonium sulphate and t-butanol thus optimized were used further for EATPP and UATPP. 2.3. Enzyme assisted three phase partitioning (EATPP) One factor at-a-time method was used to study the effect of various parameters on the extraction of FSK. Temperature and pH
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were kept constant at their optimal levels as determined experimentally (data not shown). The parameters were studied individually for StargenÒ 002 (pH 4.5, 50 °C and 2000 U/mL) and AccelleraseÒ 1500 (pH 4.5, 50 °C and 1500 FPU/mL). One unit of enzyme activity was defined as the amount of enzyme that would liberate one mole of reducing sugar per minute under optimized assay conditions. StargenÒ 002 is a blend of a-amylase and glucoamylase which hydrolyses starch. AccelleraseÒ 1500 is a mixture of cellulase and glucosidase having action on cellulose. In EATPP, enzyme pretreatment was given to C. forskohlii root slurry prior to TPP using StargenÒ 002 and AccelleraseÒ 1500 in Mcllvaine’s buffer. Specific amount of enzyme was added to the slurry, and the slurry was incubated with gentle stirring with a magnetic stirrer for 1 h. After incubation, FSK was extracted by TPP using the optimized conditions as determined earlier, and the extract was quantified for FSK. For StargenÒ 002 and AccelleraseÒ 1500 pretreatment, enzyme concentration (16–80 U/g of substrate and 30–150 FPU/g of substrate, respectively) were optimized. Effect of combination of these two enzymes was also studied. For this, slurries were incubated for 1 h after addition of StargenÒ 002 and AccelleraseÒ 1500 (64 U/g of substrate and 90 FPU/g of substrate, respectively) at pH 4.5, other conditions of extraction by EATPP being the same. Slurries were prepared in Mcllvaine’s buffer system [26]. Incubation time of combined StargenÒ 002 and AccelleraseÒ 1500 pretreatment was optimized by incubating the slurries (pH-4.5, 50 °C) for 30–120 min. The extract so obtained was quantified for FSK content. 2.4. Ultrasound assisted three phase partitioning (UATPP) In UATPP, ultrasound pretreatment was given to C. forskohlii root slurry prior to TPP. Optimization was carried out by using one factor at-a-time method. Optimization of power requirement for the maximum extraction of FSK after TPP was done by varying the power from 18 to 58 W for 10 min. Effect of duty cycle (30–70%) as a pretreatment on extraction of FSK evaluated by subjecting 10% w/v of slurry prepared in distilled water at optimized power for 10 min. Further, optimization of extraction time (5–20 min) of sonication before TPP was carried out for maximum extraction of FSK. The extract was then quantified for FSK content. 2.5. Combination of ultrasonication and enzyme pretreatment followed by TPP To check the combined effect of ultrasonication and enzyme pretreatment, C. forskohlii root slurry was treated with optimized parameters obtained from ultrasonication study. Further, ultrasound treated slurry was subjected to enzyme pretreatment followed by TPP. The extract was then quantified for FSK content. 2.6. Conventional solvent extraction The C. forskohlii root powder was extracted in a Soxhlet apparatus using methanol for 12 h. The extract was cooled and then concentrated by evaporating in rotary vaccum evaporator under reduced pressure at 50 °C (Buchi Rotavapor, R-124, Switzerland). Solvent was recovered from rotavac. The FSK yield was expressed as % w/w of C. forskohlii root powder. 2.7. Analytical determination A Jasco HPLC system fitted with Zorbax eclipse XDB C18 column (5l 4.6 mm 250 mm) was used. The column was equilibrated with an acetonitrile-water (50:50) mixture as mobile phase at a flow rate of 1.5 mL/min. FSK was detected by measuring UV absorption at 217 nm [27]. Retention time of standard FSK was 9.7 min.
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The basic phenomenon of TPP is that the water/t-butanol mixture separates a crude proteinaceous extract into a protein-rich middle layer in addition to the two liquid phases. An increased activity of enzymes is often seen in this middle layer [28]. Pigments, lipids and enzyme inhibitors are concentrated in upper phase, while polar components like saccharides are enriched in the lower aqueous phase. Solvent extraction of C. forskohlii roots gave a FSK yield of 0.64 ± 0.12% w/w of substrate which was considered as 100% recovery and against which the FSK recovery by TPP was estimated. Fig. 1a–c represents the effect of ammonium sulphate loading, quantity of t-butanol and extraction time on the yield of FSK expressed as % w/w of C. forskohlii root powder, respectively. The maximum FSK yield of 0.194 ± 0.0025% w/w (corresponding to extraction efficiency of 30.31%) was achieved with 30% w/v of ammonium sulphate. Many other researchers reported 30% w/v ammonium sulphate to give best results where TPP has been used for separation of proteins/enzymes [29]. Sharma et al. [19] also found that 30% w/v of ammonium sulphate to show optimum extraction of soybean oil by TPP. Fig. 1b shows the effect of varying the ratio of t-butanol and aqueous slurry of C. forskohlii root. Decreasing the t-butanol volume by half decreased the FSK yield whereas increasing t-butanol to twice the volume did not lead to any significant increase. Thus, 1:1 ratio was employed in further studies. Optimum extraction time was found to be 60 min. FSK yield obtained was 0.197 ± 0.003% w/w with extraction efficiency of 30.83% under the optimum conditions of TPP. We prepared the slurry of root powder in distilled water (pH-7.0) and pH of the system was found to be 5.2. After addition of ammonium sulphate to the slurry, pH of the system increased to 5.5. The influence of pH on extraction yield was studied by changing the pH of the system from 4 to 7 after addition of ammonium sulphate to the slurry. The yield of FSK at different pH values (Fig. 1d) did not change significantly. Further experiments were performed under these optimum conditions of TPP without adjusting the pH of the system. 3.2. Enzyme assisted three phase partitioning (EATPP) FSK is a terpenoid which is mainly situated in cytoplasmic vesicles of roots. The root is mainly divided into outermost cork cells followed by cortex, cambium, and xylem. Cork consists of polygonal cells. Yellowish to brown masses are found in the cells of cork, cortex, medullary rays, and xylem. These are identified as cytoplasmic vesicles containing secondary metabolites/terpenoids [30]. These cytoplasmic vesicles are generally 5–12 lm in diameter and are attached to the outer wall of the cork cells by a membranous stalk. The location of FSK in such as cell structure is likely to offer some resistance to penetration by solvent before enabling its solubilization in solvents. At higher temperatures, an increased rupture of the cellular structure is possible by hydrolysis of cellulose following increased solubilization of solute into the solution which may reduce the firmness of the cell wall. It is well established that terpenoids are trapped in cytoplasmic vesicles. The cytoplasmic vesicles are present in both the bark and wood regions.
Forskolin (% w/w of substrate)
3.1. Three phase partitioning (TPP)
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3. Results and discussion
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Quantification of FSK was done by extrapolating the area under the curve to the concentration of standard FSK.
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pH Fig. 1. Optimization of parameters for three phase partitioning (a) effect of ammonium sulphate loading with t-butanol to slurry (1:1) and extraction time of 60 min, (b) ratio of t-butanol to slurry with 30% ammonium sulphate loading and extraction time of 60 min, (c) effect of extraction time with 30% ammonium sulphate loading and t-butanol to slurry (1:1), and (d) effect of pH with 30% ammonium sulphate loading, t-butanol to slurry (1:1) and extraction time of 60 min.
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Quantification of FSK in different tissues has shown terpenoids to be mainly concentrated in the woody layer [31]. Thus, it was considered worthwhile to attempt pretreatment of the C. forskohlii
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root slurry by enzymes before carrying out TPP. Aqueous extraction of oils using enzymes has been successfully used in extraction of oils from rice bran [32] and peanut [23]. EATPP has not been attempted for extraction of FSK from C. forskohlii roots so far. StargenÒ 002 enzyme contains Aspergillus kawachi alpha-amylase expressed in Trichoderma reesei and a glucoamylase from T. reesei that work synergistically to hydrolyze granular starch substrate to glucose. AccelleraseÒ 1500 is a mixture of cellulase and glucosidase that works synergistically to hydrolyze cellulosic substrate to glucose. As C. forskohlii root contained 14– 15% cellulose and 18–20% starch (determined earlier in the laboratory), these two enzymes could increase the efficiency of TPP by breaking complex cellulosic matrix that entraps FSK and thereby increase the release of FSK. Extraction of FSK carried out by varying the concentration of StargenÒ 002 showed optimum extraction of FSK at 64 U/g of substrate (Fig. 2a). Similarly, AccelleraseÒ 1500 showed maximum extraction efficiency at 90 FPU/g of substrate (Fig. 2b). With further increase in concentration, no increase in extraction efficiency was observed. Pretreatments with StargenÒ 002 and AccelleraseÒ 1500
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Time (min) Fig. 2. Optimization of parameters for enzyme assisted three phase partitioning (a) effect of StargenÒ 002 concentration with incubation time of 60 min, (b) effect of AccelleraseÒ 1500 concentration with incubation time of 60 min, (c) combined effect of enzymes with incubation time of 60 min, and (d) Effect of incubation time of combined StargenÒ 002 and AccelleraseÒ 1500.
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Table 1 Extraction of forskolin: comparison between conventional solvent extraction, UATPP, EATPP and UATPP + EATPP.
a
Parameter
Conventional solvent extractiona
UATPPa
EATPPa StargenÒ 002 + AccelleraseÒ 1500
UATPP + EATPPa
Solvent Extraction time (h) Yield of FSK (% w/w of Coleus forskohlii roots) Extraction efficiency (%)
Methanol 12 0.64 ± 0.12 –
t-Butanol 4 0.51 ± 0.01 79.95 ± 1.63
t-Butanol 4 0.53 ± 0.01 83.85 ± 2.39
t-Butanol 4 0.55 ± 0.004 85.16 ± 0.78
Results are mean ± standard deviation of three determinations.
individually gave maximum FSK yield of 0.27 ± 0.004% w/w and 0.45 ± 0.01% w/w, corresponding to extraction efficiency of 42.71 and 70.31%, respectively. Combined effect of StargenÒ 002 and AccelleraseÒ 1500 improved the yield to 0.53 ± 0.01% w/w with extraction efficiency of 83.85% (Fig. 2c). Evaluation of incubation time from 30 to 120 min showed 60 min to be sufficient for maximum extraction of FSK (Fig. 2d). Sharma et al. [32] reported that combination of protease, cellulase and amylase as a pretreatment yielded 77% recovery of rice bran oil. Extraction of peanut oil with protizyme pretreatment could increase the recovery upto 92% [23]. Shah et al. [20] reported that extraction of oil from Jatropa curcas L. with fungal proteases could increase the recovery upto 97%.
4. Conclusion Enzyme pretreatment followed by three phase partitioning is a promising approach of obtaining efficient extraction of FSK from C. forskohlii roots. Ultrasonication as a pretreatment did increase the extraction of FSK when used individually but was not very effective in further increasing the extraction of FSK when used in combination with enzyme assisted three phase partitioning. Hence, enzyme assisted three phase partitioning can be recommended for extraction of FSK. Unlike the conventional solvent extraction, which requires 12 h for complete extraction, the method developed herein takes only about 4 h for comparable yields of extractives. Acknowledgement
3.3. Ultrasound assisted three phase partitioning (UATPP) Ultrasonication has been used as a pretreatment for extraction of nutraceuticals from plants having advantages like reduced extraction time, reduced organic solvent consumption, and minimizing pollution [33,34]. Ultrasonication efficiently disintegrates bigger particles into smaller ones [35]. Hence, we first optimized this process for maximum extraction of FSK in terms of operational parameters and followed it with TPP as optimized in Section 3.1 as above. Fig. 3a–c showed a power output of 50 W, a duty cycle of 50%, and extraction time of 15 min to give maximum FSK yields of 0.368 ± 0.002% w/w, 0.46 ± 0.007% w/w, and 0.51 ± 0.01% w/w, corresponding to extraction efficiencies of 57.50%, 72.13% and 79.95%, respectively. These results are in the range of 77–88% recovery of oils from almond, apricot and rice bran, respectively, using a similar approach [24].
3.4. Combination of ultrasonication and enzyme pretreatment followed by TPP Ultrasonication with enzyme pretreatment increased the FSK yield to 0.55 ± 0.004% w/w with extraction efficiency of 85.16% (Table 1). Shah et al. [20] reported a combination of sonication and enzyme treatment with a commercial preparation of fungal proteases at pH 9.0 to give a yield of 97% oil within 2 h. A similar approach of ultrasonication followed by EATPP was followed by Gaur et al. [22] for extraction of edible oils from mango kernel, soybean and rice bran. The total time required for extraction of FSK using the combined pretreatment followed by TPP was about 4 h while conventional solvent extraction needed 12 h for similar comparable yield. FSK in the upper organic phase and middle layer was found to be 0.55 ± 0.004% w/w and 0.08 ± 0.004% w/w, respectively, while it was absent in the lower aqueous phase. Table 1 summarizes the yields of FSK obtained with TPP, EATPP, UATTP and UATTP + EATTP under the optimized conditions. It is evident that ultrasonication prior to EATPP increased the percent recovery of FSK only marginally which may not justify including this step in the extraction protocol. Hence, EATTP can be concluded to be a feasible and rapid technique for extraction of FSK. However, extensive scale-up studies are required for its use at industrial level.
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