INTRODUCTION:

Importance of Herbal Drugs and Extraction Methods:

Plants are most important source of bioactive molecules for drug discovery. Isolated bioactive molecules serve as starting materials for the various laboratory synthesis of drugs as well as a model for the production and development of biologically active compounds. Phytochemical processing of raw herbal plant materials is essentially required to optimize the concentration of known constituents and in addition to maintain their activities¹. Plants provide a continual source of medicines for humans as well as animals; they have been used since ancient times in crude forms as syrups, decoctions, liniments, infusions, powders and ointments^2,3. Natural products have been an important part of the ancient traditional medicine systems, e.g. Ayurvedic, Chinese and Egyptian⁴. Interest in utilizing natural sources in the development as well as formulation of natural skin products, as an alternative to various conventional drugs and synthetic products, contribute to rapid increase interest in research and industrial application of herbal medicinal plants⁵. According to the World Health Organization, a medicinal plant is nothing but any plant which, in one or more than one of its organs, contains substances that can be used for therapeutic purposes, or which are major precursors for chemo-pharmaceutical and herbal pharmaceutics semi synthesis. Such a plant will have its parts including leaves, fruits, rhizomes, roots, stems, flowers, barks, grains or seeds, employed in the control or treatment of a disease condition and therefore contains chemical components which are medically active. These non-nutrient herbal plant chemical compounds or bioactive components are often referred to as phytoconstituents or phytochemicals (‘phyto- ‘from Greek - phyto meaning ‘plant’)and are majorly responsible for protecting the various herbal plant against microbial infestations or infections by pests^6,7,8,9. Currently more than 80% of the world population depends on many traditional and plant derived medicine because, Plants are primarily important sources of medicines and presently about 28% of pharmaceutical prescriptions in the United States contain at least one plant-derived ingredient for final herbal formulation. In the last century, about 121 pharmaceutical products were formulated based on the more traditional knowledge obtained from various kind of sources¹⁰. In fact, it is now believed that Nature majorly contributes more than 90% to the new drug molecule. Nature has provided many of the effective agent such as dactinomycin, vinblastine, irinotecan, bleomycin, and doxorubicin, topotecan, etoposide, and paclitaxel (anticancer), amodiaquine, Mefloquine chloroquine, artemisinin, dihydro artemisinin, artemether, and arteether (antimalarial), metformin and eventually the other Biguanide, cryptolepine, Harunganin, maprouneacin (anti diabetic) Calanolide A, cucrcumin, phenethylephenoxidiol (anti-HIV drugs), isocyanate etc.^11,12. Extraction is most important step in the itinerary of phytochemical processing for the discovery of various bioactive constituents from plant materials. Selection of a suitable extraction technique is also important for the standardization of herbal products as it is important in the utilization of the removal of desirable soluble constituents, leaving out those which are not required with the aid of the solvents¹³. The development of various modern sample preparation techniques has significant advantages over conventional methods majorly in terms of reduction in organic solvent consumption as well as in minimizing various sample degradation. They also result in the removal of insoluble and undesirable components from the extract. The modern methods includesupercritical fluid extraction (SFE), microwave assisted extraction (MAE), ultrasonication assisted extraction (UAE), solid phase micro extraction (SPME) and Soxhwave, etc. Latter is a combination of Soxhlet with the source of microwaves. This combines results into rapid heating capacity of microwaves with the more simplicity of Soxhlet. In addition to that here solvent recovery is also possible and which is not the case in ordinary microwave assisted extraction. However, it has not found more widespread use yet. Classical methods are fairly standard, simpleand continue to have widespread use, but these methods can also be slow and insufficient, consume more quantities of organic solvents, and hence which results in degradation of heat labile constituents. While using conventional methods, quality related problems viz. safety, lack of consistency, and efficacy are also the issues. Furthermore, improvement in extraction efficiency, elimination of additional sample clean-up and steps before chromatographic analysis and selectivity are also the benefits of modern extraction processes¹⁴.

EXTRACTION TECHNOLOGY:

A typical extraction process may contain following steps¹⁵

1. Collection and authentication of plant material and drying

2. Size reduction

3. Extraction

4. Filtration

5. Concentration

6. Drying and reconstitution

Quality of an extract is majorly influenced by several factors such as, solvent used for extraction, plant parts used as starting material, plant material: solvent ratio, extraction procedureetc. From laboratory scale to pilot scale this all the parameters are optimized and controlled during complete extraction herbals. Various Extraction techniques separate the soluble plant metabolites through selective use of various solvents.

ADVANCED EXTRACTION METHODS:

Supercritical Fluid Extraction (SFE):

Supercritical fluid extraction is a most important modern technique used in the extraction of different plant samples. It operates based on the influence of pressure andtemperature on the supercritical liquid state. This is vital to medicinal nourishment and fields. Mostly, supercritical fluid extraction utilized carbon dioxide (CO₂₎ because of its important physical properties of moderately lower temperature and pressure conditions (7.38 MPa and 304 K respectively)¹⁶. Likewise, the yield from this important technique had been reported to be higher as compared to the steam and hydro distillation methods. Nevertheless, the major demerit of Supercritical fluid extraction is the hardware cost which restricts its utilization for profoundly mechanical parameters¹⁷. The SFE had shown higher efficiency in separating basic oils from sunflower¹⁸, apricot¹⁹, rosemary²⁰, myrtle²¹, palm²², almond²³, juniperus²⁴, soybeans²⁵, jojoba²⁶, sesame²⁷, pistachio²⁸, celery²⁹, parsley³⁰.

The extraction is carried out in big high-pressure equipment in continuous or batch manner. In both cases, the supercritical solvent is get put in contact with the different kinds of material from which a desirable product is to be separated or extracted. Generally cylindrical big extraction vessels are used for different sample preparation¹⁵. In batch processing solid material is placed into extraction vessel and then the supercritical solvent is fed in until the final target extraction conditions are reached. And supercritical solvent is fed continuously in semi batch processing, through a high pressure pump at a fixed flow rate, to precipitate solute from supercritical solution one or more than one separation stages are used. Supercritical fluid technology is now recognized as amost effective extraction technique with efficiency comparable to existing chemical analysis methods. Supercritical fluid extraction is favourably applicable for the quantitative and qualitative identification of constituents of natural products, including different heat-labile compounds³¹. Several solvents can be used for Supercritical fluid extraction, such as, pentane, hexane, butane, sulfur hexafluoride, nitrous oxide and fluorinated hydrocarbons³². Carbon dioxide (CO₂₎ is the most commonly used extraction solvent in Supercritical fluid extraction¹⁵. CO₂ alone is non selective but its selectivity and capacity of extraction can be improved or increased by using a co solvent or modifier. Co-solvent can easily remove after the complete extraction of herbals. CO₂ is generally the most desirable solvent in Supercritical fluid extraction, because its critical temperature is only 304 K, which makes it more attractive for the extraction of various heat–labile compounds. In addition, CO₂ issafe (non-flammable, non-explosive), an inert, odorless, colorless, inexpensive, noncorrosive, clean solvent that results into no leaving of solvent residue in the product; it is also non-toxic and is mainly accepted as a harmless ingredient in pharmaceuticals and food industry, and it is easily removable from the extracted oil by very simple expansion. Moreover, carbon dioxide has a viscosity, low surface tension and high diffusivity which make it more attractive as a supercritical solvent^15,33.

There are many other liquids and gases that are highly efficient as extraction solvents when put under different pressure condition³⁴.

a. Coupled SFE-SFC:

System in which a sample to be extracted is extracted with a supercritical solvent which then places the extracted material in the inlet part of a supercritical fluid chromatographic system. The extract is than chromatographed directly using important supercritical fluid.

b. Coupled SFE-GC and SFE-LC:

System in which a sample is extracted using a supercritical fluid which is then mainly depressurized to deposit the extracted material in the inlet part or a column of gas or different liquid chromatographic system respectively. Supercritical fluid extraction is characterized by reliability, robustness of sample preparation, high yield, less time consuming and also has potential for coupling with number of various chromatographic methods.

Advantages of Supercritical fluid extraction over conventional methods can be summarized as follows^15,35

1. Extraction of constituents at low temperature, which avoids damage of constituents from heat

2. Better diffusivity

3. No solvents residue

4. Fast extraction

5. Environment friendly

6. Low viscosity of supercritical fluid, which allow more selective extractions

Microwave-assisted extraction (MAE):

It simply termed as microwave extraction because that combines microwave and traditional solvent extraction together. Heating the plant tissue and solvents using microwave increases the kinetic of extraction, hence it iscalled as microwave-assisted extraction³⁶. The target for heating in dried plant material is nothing but minute microscopic traces of moisture that mainly occurs in plant cells. The heating up of this moisture inside the plant cell due to microwave effect, results in the evaporation and then generates tremendous pressure on the cell wall. The cell wall is pushed from inside due to the pressure and then the cell wall gets ruptures. Thus the exudation of active constituents from the ruptured cells occurs, and hence increasing the yield of herbal; phytoconstituents^37,38. Upon absorption by a different material, electromagnetic energy of microwaves is converted to the heat energy. 2.45 GHz (2450 MHz) is the most commonly used frequency for commercial microwave instruments, which has an energy output of about 600-700 W³⁹. Microwave-assisted extraction is environment friendly, simple and economical technique for the extraction of biologically active compounds from the different plant materials⁴⁰. Samra et. al. had first time used microwave domestic ovens in 1975 for the treatment of various biological samples for metal analysis⁴¹. In 1986 the application of Microwave-assisted extraction for plant materials was first reported by Ganzler and co-workers⁴². The application of Microwave-assisted extraction provides many advantages, such as decreasing the thermal degradation, increasing the extract yield and selective heating of vegetal material. Microwave-assisted extraction is also regraded as a green technology because it reduces the usage of different organic solvent. There are two types of Microwave-assisted extractionmethods: solvent extraction (usually for non-volatile compounds) and solvent-free extraction (usually for volatile compounds)^43,44.

Ultrasonic-assisted extraction (UAE):

Ultrasonic-assisted extraction, also called ultrasonic sonication or extraction, which uses ultrasonic wave energy in the carrying out of extraction. Ultrasound in the solvent producing cavitation which accelerates the diffusion and dissolution of the solute as well as the heat transfer, which mainly improves the extraction efficiency of complete extraction. The other advantage of Ultrasonic-assisted extraction includes low energy and solvent consumption, and the reduction of extraction time and temperature. Ultrasonic-assisted extraction is applicable for the extraction of unstable and thermolabile compounds. Ultrasonic-assisted extraction is commonly employed in the extraction of different types of natural products^45,46. Ultrasonic-assisted extraction involves application of high-frequencyand high-intensity sound waves and their interaction with materials. Ultrasonic-assisted extractionis a most useful technology as it does not require complex instruments. and is relatively low-cost. It can be used both on small as well as large scale⁴⁷. Several probable mechanisms for ultrasonic enhancement of extraction, such as improved penetration, cell disruption, and enhanced capillary effect, swelling and hydration process have been proposed⁴⁸. If in a liquid the intensity of ultrasound is increased, then it reaches at a point where the intramolecular forces are not able to hold the molecular structure intact, so it breaks down and bubbles are created, this process is called cavitation⁴⁹.

Soxhlet extraction:

The Soxhlet extraction method integrates the advantages of the percolation and reflux extraction, which utilizes the principle of siphoning and reflux to continuously extract the herb with fresh solvent. The Soxhlet extraction is an automatic as well as continuous extraction method with high extraction efficiency which requires less solvent consumption and time than percolation or maceration. The high temperature and long extraction time in the Soxhlet extraction will results in increase the possibilities of thermal degradation⁵⁰. The degradation of catechins in tea was also observed in Soxhlet extraction due to the application of high extraction temperature. The concentrations of bothtotal alkaloids and total polyphenols from the Soxhlet extraction method at 70°C decreased compared to those from the maceration method which applied under 40 °C^51,52. In this method, finely ground sample is placed in a “thimble” or porous bagwhich made froma cellulose or strong filter paper, which is place, is in thimble chamber of the Soxhlet apparatus. Primarily Extraction solvents is heated in the bottom flask, vaporizes into the sample thimble, then condenses in the condenser and drip back. When the liquid content reaches the siphon arm, then again the liquid contents emptied into the bottom flask and the process is continued. This method requires a smaller quantity of solvent as compared to maceration⁵³. However, the Soxhlet extraction comes with disadvantage such as exposure to flammable and hazardous liquid organic solvents, with potential toxic emissions during extraction. Solvents which used in the extraction system need to be of high-purity that might add to cost. This procedure is may contribute to pollution problem and may not environmental friendly, compared to advance extraction method such as supercritical fluid extraction (SFE)⁵⁴. The ideal sample for Soxhlet extraction is also limited to a finely divided and dry solid⁵⁵ and many factors such as solvent-sample ratio, temperature and agitation speed need to be considered for this extraction method⁵⁶.

CONCLUSION:

All stages of extractions, from the pre-extraction to the complete extraction are equally important in the study of medicinal plants as well as in the herbal formulation. The sample preparation such as grinding and drying affected the phytochemical constituent’s and efficiency of the final extractions; that eventually have an effect on the final extracts. Various kinds ofNatural products have a contributed to drug development over the past few decades and continue to do so. The time-consuming and lab-intensive of extraction and isolation processes, however, have hindered the application of natural products in drug development as well as herbal formulation. As technology continues to develop, different kinds of more and more new rapid and automatic techniques have been created to separate and extract natural products, which might reach the requirement of high-throughput screening.The modern extraction methods, also regarded as green extraction methods, including supercritical fluid extraction (SFE), microwave assisted extraction (MAE), ultrasonication assisted extraction (UAE), and Soxhlet extraction, have also been the subject of increased attention in recent years due to their selectivity, high extraction yields, stability of the target extracts and process safety merits. Some of those green methods have become routine sample preparation methods for analytical purposes. In conclusion, there isincreasing and a clear interest in the isolation and extraction of natural products, herbal formulations and their advantageous applications.

REFERENCES:

1. Aziz, R.A., Sarmidi, M.R., Kumaresan, S., 2003. Phytocehemical processing: the next emerging field in chemical engineering aspects and opportunities. J. Kejurut. Kim. Malay. 3, 45–60.

2. Saad B, Azaizeh H, Said O. Tradition and perspectives of Arab herbal medicine: a review. Evid Based Complement Alternat Med. 2005;2: 475–9.

3. Ghorbani A. Clinical and experimental studies on polyherbal formulations for diabetes: current status and future prospective. J Integr Med. 2014;12: 336–45.

4. Sarker, S.D. andNahar, L. (2007). Chemistry for Pharmacy Students General, Organic and Natural Product Chemistry. England: John Wiley and Sons. pp 283-359.

5. Mukherjee PK, Maity N, Nema NK, Sarkar BK (2011) Bioactive compounds from natural resources against skin aging. Phytomedicine 19: 64-73.

6. Abo, K.A.; Ogunleye, V.O. andAshidi JS (1991). Antimicrobial poteintial of Spondiasmombin, Croton zambesicusand Zygotritoniacrocea. Journal of Pharmacological Research. 5(13): 494-497.

7. Liu, R.H. (2004). Potential synergy of phytochemicals in cancer prevention: mechanism of Action. Journal of Nutrition. 134(12 Suppl):3479S-3485S.

8. Doughari, J.H.; Human, I.S, Bennade, S. andNdakidemi, P.A. (2009). Phytochemicals as chemotherapeutic agents and antioxidants: Possible solution to the control of antibiotic resistant verocytotoxin producing bacteria. Journal of Medicinal Plants Research. 3(11): 839-848.

9. Nweze, E.L.; Okafor, J.L. andNjoku O (2004). Antimicrobial Activityies of Methanolic extracts of Trumeguineesis (Scchumn and Thorn) and Morindalucindaused in Nigerian Herbal Medicinal practice. Journal of Biological Research and Biotechnology. 2(1): 34-46.

10. Verma S and Singh SP, Current and future status of herbal medicines, Veterinary World, 2008; 1(11): 347-350.

11. Moshi MJ, Current and future prospects of integrating traditional and alternative medicine in the management of diseases in Tanzania, Tanzania Health Research Bulletin, 2005; 7(3):159-166.

12. Maria Russo, Carmela Spagnuolo, Idolo Tedesco and Gian Luigi Russo, Phytochemicals in Cancer prevention and therapy: Truth or Dare, Toxins, 2010; 2(4): 517-551.

13. Co, M., Fagerlund, A., Engman, L., Sunnerheim, K., Sjoberg, P.J.R., Turner, C., 2012. Extraction of antioxidants from Spruce (Piceaabies) bark using ecofriendly solvents. Phytochem. Anal. 23, 1– 11.

14. Kothari V, Pathan S, Seshadri S. 2010. Antioxidant activity of M. zapota and C. limon seeds. J. Nat. Remedies. 10[2]: 175-180.

15. Handa SS, Khanuja SPS, Longo G, and Rakesh DD. 2008. Extraction technologies for medicinal and aromatic plants. Trieste: ICS UNIDO.

16. Carvalho, R.N., Moura, L.S., Rosa, P.T.V., and Meireles, M.A.A., 2005, Supercritical fluid extraction from rosemary (Rosmarinusofficinalis): Kinetic data, extract's global yield, composition, and antioxidant activity, J. Supercrit. Fluids, 35 (3) 197–204.

17. Meireles, M.A.A., 2003, Supercritical extraction from solid: process design data (2001–2003), Curr. Opin. Solid State Mater. Sci., 7 (4-5), 321–330.

18. Calvo, L., Cocero, M., and Díez, J., 1994, Oxidative stability of sunflower oil extracted with supercritical carbon dioxide, J. Am. Oil Chem. Soc., 71 (11), 1251– 1254.

19. Özkal, S., Yener, M., and Bayındırlı, L., 2005, Response surfaces of apricot kernel oil yield in supercritical carbon dioxide, LWT Food Sci. Technol., 38 (6), 611–616.

20. Zermane, A., Meniai, A.H., and Barth, D., 2010, Supercritical CO₂ extraction of essential oil from Algerian rosemary (Rosmarinusofficinalis L.), Chem. Eng. Technol., 33 (3), 489–498.

21. Zermane, A., Larkeche, O., Meniai, A.H., Crampon, C., and Badens, E., 2014, Optimization of essential oil supercritical extraction from Algerian Myrtuscommunis L. leaves using response surface methodology, J. Supercrit. Fluids, 85, 89–94.

22. MdZaidul, I.S., Nik Norulaini, N.A., and Mohd Omar, A.K., 2006, Separation/fractionation of triglycerides in terms of fatty acid constituents in palm kernel oil using supercritical CO₂, J. Sci. Food Agric., 86 (7), 1138–1145.

23. Marrone, C., Poletto, M., Reverchon, E., Stassi, A., 1998, Almond oil extraction by supercritical CO₂: Experiments and modelling, Chem. Eng. Sci., 53 (21), 3711–3718.

24. Larkeche, O., Zermane, A., Meniai, A.H., Crampon, C., and Badens, E., 2015, Supercritical extraction of essential oil from Juniperus communis L. needles: Application of response surface methodology, J. Supercrit. Fluids, 99, 8–14.

25. Reverchon, E., and Osséo, L.S., 1994, Comparison of processes for the supercritical carbon dioxide extraction of oil from soybean seeds, J. Am. Oil Chem. Soc., 71 (9), 1007–1012.

26. Salgın, U., 2007, Extraction of jojoba seed oil using supercritical CO₂+ethanol mixture in green and high-tech separation process, J. Supercrit. Fluids, 39 (3), 330–337.

27. Xu, J., Chen, S., and Hu, Q., 2005, Antioxidant activity of brown pigment and extracts from black sesame seed (Sesamumindicum L.), Food Chem., 91 (1), 79–83.

28. Palazoglu, T.K., and Balaban, M.O., 1998, Supercritical CO₂ extraction of lipids from roasted pistachio nuts, Trans. ASAE, 41 (3), 679–684.

29. Marrone, C., Poletto, M., Reverchon, E., Stassi, A., 1998, Almond oil extraction by supercritical CO₂: Experiments and modelling, Chem. Eng. Sci., 53 (21), 3711–3718.

30. Louli, V., Folas, G., Voutsas, E., and Magoulas, K., 2004, Extraction of parsley seed oil by supercritical CO₂, J. Supercrit. Fluids, 30 (2), 163–174.

31. Mohameda, R.S., Mansoor, G.A. 2002. The use of supercritical fluid extraction technology in food processing. Food Technol Magazine. The World Markets Research Centre, London, UK.

32. Reverchon E, Marco I. 2006. Supercritical fluid extraction and fractionation of natural matter. J. Supercrit Fluids. 38: 146-166.

33. Tonthubthimthong P, Chuaprasert S, Douglas P, Luewisutthichat W. 2001. Supercritical CO2 extraction of nimbin from neem seeds an experimental study. J. Food Eng. 47: 289-293.

34. Patil, P.S. andShettigar, R. (2010). An advancement of analytical techniques in herbal research J. Adv. Sci. Res. 1(1); 08-14.

35. Ahuja, S., and Diehl, D. 2006. Sampling and Sample prepration. In: Comprehensive Analytical Chemistry, Vol. 47 (Eds.), S Ahuja, and N Jespersen, Oxford, UK: Elsevier (Wilson and Wilson) Chap-2, pp.15-40.

36. Delazar A, Nahar L, Hamedeyazdan S, Sarker SD. Microwave-assisted extraction in natural products isolation. Methods Mol Biol. 2012; 864:89-115

37. Gordy WWV, Smith RF Trambarulo. Microwave Spectroscopy. Wiley, New York. 1953.

38. Goldman R. Ultrasonic Technology. Van Nostrand Reinhold, New York. 1962.

39. Jain T, Jain V, Panday R, Vyas A, Shukla SS. 2009. Microwave assisted extraction for phytoconstituents - An overview. Asian J. Res. Chem. 2: 19-25.

40. Hemwimon S, Pavasant P, Shotipruk A. 2007. Microwave assisted extraction of antioxidativearthraquinones from roots of Morindacitrifolia. Sep. Purif. Technol. 54: 44-50.

41. Letellier M, Budzinski H. 1999. Microwave assisted extraction of organic compounds. EDP Sciences, Wiley-VCH, Analysis. 27: 251-271.

42. Kaufmann BA, Christen P. 2002. Recent extraction techniques for natural products: microwaveassisted extraction and pressurized solvent extraction. Phytochem. Anal. 13: 105-113.

43. Chemat F, Cravotto G. Microwave‑assisted extraction for bioactive compounds. Boston: Springer; 2013.

44. Vinatoru M, Mason TJ, Calinescu I. Ultrasonically assisted extraction (UAE) and microwave assisted extraction (MAE) of functional compounds from plant materials. TrAC Trends Anal Chem. 2017;97: 159–78

45. Barba FJ, Zhu Z, Koubaa M, Sant’Ana AS, Orlien V. Green alternative methods for the extraction of antioxidant bioactive compounds from winery wastes and by‑products: a review. Trends Food Sci Technol. 2016;49: 96–109.

46. Chemat F, Rombaut N, Sicaire AG, Meullemiestre A, Fabiano‑Tixier AS, Abert‑Vian M. Ultrasound assisted extraction of food and natural products. Mechanisms, techniques, combinations, protocols and applications. A review. UltrasonSonochem. 2017;34: 540–60.

47. Dai J, Mumper RJ. 2010. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules. 15: 7313-7352.

48. Huaneng X, Yingxin Z, Chaohong H. 2007. Ultrasonically assisted extraction of isoflavones from stem of PuerariaLobata (Willd.) Ohwi and its mathematical model. Chin J. Chem. Eng. 15: 861-867.

49. Baig S, Farooq R, Rehman F. 2010. Sonochemistry and its industrial applications. World Appl. Sci. J. 10: 936-944.

50. Wei Q, Yang GW, Wang XJ, Hu XX, Chen L. The study on optimization of Soxhlet extraction process for ursolic acid from Cynomorium. ShipinYanjiu Yu Kaifa. 2013;34(7):85–8.

51. Chin FS, Chong KP, Markus A, Wong NK. Tea polyphenols and alkaloids content using soxhlet and direct extraction methods. World J Agric Sci. 2013;9(3):266–70.

52. Xu FX, Yuan C, Wan JB, Yan R, Hu H, Li SP, Zhang QW. A novel strategy for rapid quantification of 20(S)‑protopanaxatriol and 20(S)‑protopanaxadiolsaponins in Panaxnotoginseng, P. ginseng and P. quinquefolium. Nat Prod Res. 2015;29(1):46–52.

53. Handa SS, Khanuja SPS, Longo G, Rakesh DD (2008) Extraction Technologies for Medicinal and Aromatic Plants, (1stedn), no. 66. Italy: United Nations Industrial Development Organization and the International Centre for Science and High Technology.

54. Naudé Y, De Beer WHJ, Jooste S, Van Der Merwe L, Van Rensburg SJ (1998)Comparison of supercritical fluid extraction and Soxhlet extraction for the determination of DDT, DDD and DDE in sediment. Water SA 24: 205-214.

55. Methods Optimization in Accelerated Solvent Extraction in Technical note (2013) 208: 1-4.

56. Amid, Salim RJ, Adenan M (2010) The Factors Affecting the Extraction Conditions for Newroprotective Activity of Centellaasiatica evaluated by Metal Chelating Activity Assay. J ApplSci 10: 837-842.

Received on 30.03.2020 Modified on 17.06.2020

Asian J. Res. Pharm. Sci. 2020; 10(4):287-292.

DOI: 10.5958/2231-5659.2020.00050.8