Pharmaceutical Standardization of Guggulu Śodhana

Vyas, Shukla, Ruknuddin, and Prajapati: Pharmaceutical Standardization of Guggulu Śodhana

Authors

INTRODUCTION

Āyurvedic pharmaceutics is enriched by various processes and techniques. Śodhana (preliminary processing techniques), Mārana (incineration), Bhāvana (levigation), Mardana (grinding) etc. procedures are described in classics to achieve perfect formulation composition and to get desired efficacy. Among them, Śodhana is a preliminary process that helps in eliminating possible physical or chemical blemishes from raw material and making them suitable for therapeutic application .[1] Guggulu (Commiphora wightii, (Arn) Bhandari) is a well known herbal drug, which is being used in vast range of diseases since Vedic period. It is advocated in Kuṣṭha (skin diseases), Medoroga (lipid disorders), Āmavāta (rheumatoid arthritis), Sandhigatavāta (osteoarthritis), Gulma (Abdominal lump), Śotha (odema) etc.[2]

Guggulu is also popular as dietary supplement to reduce cholesterol.[3] Many modern pharmaceutics prepare Guggulu formulations by using Guggulu in extract form. Guggulu is gaining its attention in world market nowadays.[4] Around 90 tonnes per annum of Guggulu extract powder is demanded in Indian market.[5] To meet increased market demand, many Āyurvedic pharmacies are preparing these formulations on large scale. The exudate of this plant is therapeutically active and need to be processed by following classical guidelines before its utilization in therapeutics.

Some studies reported adverse effects like skin rashes, irregular menstruation, diarrhoea, headache, mild nausea, eructation, hiccough, and with very high doses, liver toxicity too.[6] On the other hand, studies reported reduced Gastric irritancy[7] and increased pharmacological action with Śodhita Guggulu.[8] But, unfortunately, no standard manufacturing procedure of Guggulu Śodhana is available till date. Considering this, it has been attempted to develop standard manufacturing procedure of Guggulu Śodhana of laboratory scale.

Various Guggulu Śodhana medias are described in Āyurveda like Godughdha, Gomūtra, Triphalā Kvātha, Gudūchῑ Kvātha etc.[9-10] Gomūtra is dominated by kṣārῑya (alkaline) substances, kaṭu-tῑkta rasa, tῑkṣṇa and laghu guṇa, uṣṇa vῑrya and kaṭu vipāka. Gomūtra is also reported to have bio-enhancer,[11] anti diabetic, anti oxidant,[12] and anti cancer[13] activities. Research works regarding Guggulu Śodhana are less in number.[14] Only one work is found on standardization Guggulu Śodhana in Triphalā Kaṣāya,[15] but no work is reported on Standardization of Guggulu Śodhana in presence of Gomūtra till date. Considering this, it is planned to develop pharmaceutical standardization of Guggulu Śodhana.

MATERIALS AND METHODS

Collection of drug: Raw Guggulu [Commiphora wightii (Arn) Bhandari.] was procured from Gujarat State Forest Department Corp. Ltd., Vadodara during Feb 2012 (Batch no. B 01, Code-128500). Fresh Gomūtra was collected locally from Jamnagar.

Pharmaceutical evaluation

Śodhana procedure: Guggulu Śodhana was done by Parῑṣravaṇa (dissolving and filtering) method.[16] External impurities like stone, bark, wood etc. from raw material were removed manually. Raw Guggulu was made into small pieces. Four litres of Gomūtra was added to 1 kg of Aśudhdha Guggulu (AG) in a stainless steel vessel. Proportion of Gomūtra and Guggulu was decided as per the reference of Bṛhat Rasarāja Suṇdara.[17] The contents were subjected to mild heat maintaining temperature between 70-85°C to facilitate dissolution of Guggulu. After complete dissolution, the contents were filtered through cotton cloth (madarpat fabric) in hot condition. Contents remained as residue in cloth were discarded. The filtrate was subjected to further heating at 70°C till complete evaporation of liquid. Obtained semisolid mass was shifted to ghee smeared stainless steel trays, spread into thin uniform layers and dried in under sun. The dried mass was carefully collected as Śudhdha Guggulu (SG) and stored (Fig. 1).

Figure 1

Guggulu Śodhana Procedure

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Physico-chemical evaluation

Physico-chemical parameters like loss on drying, ash value, acid insoluble ash, water soluble extractives, methanol soluble extractives, pH and volatile oil content were carried out by following standard procedures.[18]

High Performance Thin Layer Chromatography (HPTLC): For HPTLC study, 5 g of drug was extracted with methanol by soxlate extract method. It was then combined with methanol to adjust the volume to 25 ml. A CAMAG (Switzerland, version 1.2.1)) HPTLC system equipped with a sample applicator Linomat V was used for application of samples. CAMAG TLC Scanner 3, Reprostar and Wincats 4.02 were used for scanning the plates. CAMAG twin through glass chamber was used for developing the plates. Pre-coated silica gel GF 254 plate was used as stationary phase. Petroleum ether (60-80°C): Ethyl acetate: Methanol (6:2:0.5) v/v was used as mobile phase as per reference of ICMR database.[19] After 30 minutes of chamber saturation, plate was developed, and then scanned under short UV (254 nm) and long UV (366 nm) and thereafter sprayed with Vaniline-sulphuric acid reagent for color reaction.

High Performance Liquid Chromatography (HPLC): Chromatographic conditions for estimation of Guggulsterone-E and Guggulsterone-Z through HPLC are Column: ODS (Octadecasaline) C 18(2), 5 microne size, 250×40 mm (Merck) RP-18 Lichrocart 250-4; Detection: SPD-20A prominence UV- Visible detector, Wavelength detection: 242 nm; Mobile Phase: Water:Acetonitrile (55:45); Temperature: Room temperature; Flow Rate : 1 ml/min; Pressure: 153 kgf; Note : 47.6 mg sample in 50 ml Acetonitrile.

Fourier transform infrared spectroscopy (FTIR): FTIR spectrometer specifications are as follows: Instrument: Nicolet Instrument Corporation, USA, MAGNA 550; Method: KBr pallet, Ratio (100mg KBr:1mg Sample) Range: 4000 to 50 cm-1Spectral Resolution: 0.4cm-1

Microbial overload: Culture medium was prepared by following method: As per requirement weighed solid sample was dissolved in appropriate distilled water and agar (Mac conkey Agar for bacteria and Sabroud’s for yeast and mould) was added. The solution was heated and final volume was made. The medium was distributed in flasks and sterilized by autoclaving at 121°C for 15 min. In the sterilized area, the solution was poured into plates and kept for cooling. After that, weighed sample spread on plates in sterilized area. Plates were kept downwards. Plates were observed after 24 h for bacteria and 36 to 48 h for yeast and moulds.

Heavy metal content test (ICP - OES): Heavy metal analysis was done by ICP–OES (Inductive Coupled Plasma – Optical Emission Spectrometer) method. [Make : Perkin Elmer; Model : Optima 3300 RL] Sample Preparation : For acid digestion of sample, take 0.25 g sample and add 5ml of HCl + 5ml of HNO3+ 1ml H2O2 in a closed device using temperature control microwave heating at 200° C for 15 minutes then after cooling vessel device, solution filter it and wash by de-ionized water and make up 25ml solution. Instrument calibrated with reference standard 100 ppm.

RESULTS AND DISCUSSION

Guggulu Śodhana was done in 10 batches; the average details of which are shown in Table 1. Average 80.56% yield was observed during Guggulu Śodhana. To check the percentage of Gomūtra in each sample of Śodhita Guggulu, attempts were made to calculate solid contents of Gomūtra individually, which was found to be 4.6 at an average.

Guggulu Śodhana is described mainly by two methods in classics. In one method, Dolayantra Svedana (boiling) is advised in pottalῑ[20] and in another method, Svedana by Parῑṣravaṇa method.[16] Pilot batches were attempted by following these two methods before commencing the actual pharmaceutical procedure. Excessive loss and difficulty in drying was noticed in first batch done by following Svedana method. In this procedure, Gomūtra was added repeatedly in increments to keep Pottalῑ immersed completely. Thirteen litres of Gomūtra was required to complete the processing of 0.5kg Guggulu. Squeezing of Pottalῑ was also done repeatedly to avoid blocking of cloth pores by Guggulu gum-resin. For obtaining maximum yield, Svedana was continued for three days. The contents obtained at the end of boiling were subjected for drying in sun rays and later in hot air oven. But, complete drying was not observed and the contents remained semi liquid in consistency. Due to these practical difficulties, method of Parῑṣravaṇa was adopted in the second pilot batch.

Table 1

Results of Śodhita Guggulu

Batch No.Wt of Guggulu (g)Yield
(%)
Loss
(g)
Loss
(%)
Before
Śodhana
After
Śodhana
Residue(g)
1100075575.5245↓24.5239
2100085485.4146↓14.6140
3100078078.0220↓22.0212
4100080880.8192↓19.2185
5100077677.6224↓22.4218
6100074574.5225↓22.5248
7100087687.6124↓12.4118
8100080780.7193↓19.3188
9100079379.3207↓20.7199
10100086286.2138↓13.8132
Average1000805.680.56194.419.44187.9

↓=loss

As Guggulu is plant exudates, it contains impurities like pieces of stem bark, thorn, leaves etc. The percentage of these foreign matters should not be exceeded to 4%.[21] These physical impurities were removed manually from Aśuddha Guggulu and found negligible in amount. Aśuddha Guggulu was crushed in small pieces which facilitates better and easy solubility in Gomūtra. Guggulu was dissolved in Gomūtra by heating and maintaining the temperature in between 75-80°C for 3 hours. (Table-2)

Table 2

Average time and duration of process

Process DurationTemp
HeatingBefore filtering3 hrs75-80°C
After filtering4 hrs65-70°C
DryingSun rays9 hrs28-32°C
DryingSun rays9 hrs28-32°C
DryingSun rays9 hrs28-32°C

Amber yellow colour of Gomūtra turned to creamish - yellow after dissolution of Guggulu and mixed smell of Gomūtra and Guggulu was perceived. After 3 hours of continuous heating, almost all Guggulu got dissolved. During heating, mixed smell of Gomutra and Guggulu was perceived. After complete dissolution, solution became further sticky and the contents were squeezed through the cotton cloth in hot condition. The residual part in cotton cloth containing physical and insoluble impurities was discarded that is whitish and rubbery, may be a part of resin. Filtrate was further heated to evaporate the moisture content. During this process, temperature was maintained between 65-70°C. More heat leads to excessive frothing and spilling of the material. Hence, large sized containers should be used. In the evaporating stage, intense (ammonic) smell, and irritation of eyes was perceived that may be due to liberation of ammonia present in Gomūtra. Continuous stirring was needed in last stages to avoid sticking and burning of the material. After obtaining semi-solid consistency, mass was shifted into stainless steel trays. At this stage, Guggulu was very sticky, hence to avoid loss and for easy collection tray was smeared with ghee. After sun drying, the material become harder and colour turned to dark brown. As this method is convenient; further followed in 10 batches to develop standard manufacturing methods of 1 kg each of Aśuddha Guggulu and 4 l of Gomūtra. Average 7 h was the time duration required for dissolving and evaporation procedure. For sun drying it took 3 days. An average of 80.56% yield was obtained. Average 187.9 g residue was found.

In Organoleptic evaluation, Yellowish brown colour of AG was turned to dark brown after Śodhana in Gomūtra. Significant balsamic odor was perceived in Raw Guggulu sample while after Śodhana, mixed smell of Gomūtra and Guggulu was perceived in SG. Taste of AG is bitter- astringent while SG has bitter taste. Guggulu became stickier after Śodhana due to Gomūtra.

Physico-chemical parameters of Guggulu samples were given in Table 3. All parameters of Aśodhita Guggulu samples were found within the limits given by API.[21] The pH of Śodhita sample was increased to 6 from 5. Gomūtra was used as Śodhana media, which is alkaline (pH=7.40) in nature may contribute to increase in pH. Loss on drying was found more (15.33%) in SG in comparison to AG indicating presence of more moisture content in Śodhita sample. This loss in weight in the Śodhita samples, would be due to Gomūtra used in Śodhana.Gomūtra consist a lot of Kṣariya substances which are hygroscopic in nature. SG had more Ash value (12.69%) than AG. It may be due to some inorganic substances incorporated during Śodhana procedure. Water soluble extractive was found more in SG. It may be because of Śodhana in Gomūtra that extracted some aqueous principles. Methanol soluble extractive was found decreased after Śodhana procedure. No significant Changes were observed in volatile oil values of Aśudhdha and Śodhita Guggulu. It may because content in Gomūtra was clutched the volatile oil of Guggulu.

Table 3

Physico-chemical parameters of Aśuddha and Śuddha Guggulu

ParametersAPI For plain GugguluAGSG
pH-5.06.0
Loss on drying (1100°C)%Not>1412.7615.33
Ash value (w/w)%Not>54.7312.69
Acid insoluble ash (w/w)%Not>12.303.90
Water soluble extractive (w/w)%Not>5345.6563.86
Methanol soluble extractive (w/w)%Not>2738.8325.49
Volatile oil (w/w)%Not>10.990.97

In HPTLC study, methanolic extract of AG showed 13 numbers of spots at 254 nm wavelength and 11 spots at 366 nm wavelength and SG showed 12 numbers of spots at 254 nm wavelength and 10 spots at 366 nm wavelength (Table 4 and Fig. 2). Variable number of spots was found in different groups. In Spectral comparison, common component at 0.05, 0.10, 0.43, 0.55, 0.67, 0.77, 0.88 and 0.97 Rf was found in both Guggulu samples.

In HPLC study, marker compound was estimated (Table-5) Guggulsterone-E was found more (0.326%) in AG, while it was 0.215% in SG. Simultaneously Guggulsterone −Z was found more (0.722%) in AG and in SG it was 0.567%. HPLC profile of both Guggulu samples showed decreased concentration of Gugulsterone-E and Z after Śodhana of Guggulu. The active components of the plant are the Guggulsterones, specifically the stereoisomers, Guggulsterone E and Guggulsterone Z.[22] It is found to be responsible for lowering blood lipids.[23] Concentration of bio active component are influenced by many factors like Season, collection area , annual rain fall, geographical variation, Planting, harvesting practices, solvent system, mobile phase etc.[24] Impact of reduction of these sterones after Śodhana is not justifiable here and needs more data from experimental and clinical trials.

Figure 2

HPTLC profile of Aśuddha and Śuddha Guggulu

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Table 4

HPTLC profile of Aśuddha and Śuddha Guggulu

No.Samples 254 nm 366 nm
  No. of spotsRfNo. of spotsRf
1AG130.04, 0.10, 0.24, 0.33, 0.36, 0.44, 0.55, 0.67, 0.77, 0.84, 0.91, 0.96, 0.99110.04, 0.10, 0.14, 0.24, 0.36, 0.44, 0.61, 0.72, 0.82, 0.88, 0.99
2SG120.05, 0.09, 0.17, 0.21, 0.31, 0.39, 0.43, 0.55, 0.67, 0.79, 0.93, 0.97100.05, 0.09, 0.18, 0.21, 0.34, 0.49, 0.59, 0.71, 0.88
Table 5

HPTLC profile of Aśuddha and Śuddha Guggulu

No.Sample nameGuggulsteron-E (%w/w)Guggulsteron-Z (%w/w)
1AG0.3260.722
2SG0.2150.567

FTIR evaluation

In FTIR study, observed frequencies are given in Table-6 and 7. AG has alkanes, alcohol & phenols, amines, aldehydes & ketones, carboxylic acids & derivatives, sulphur, phosphorous and silicon fictional groups, whereas SG has alkanes, alcohol & phenols, carboxylic acids & derivatives, sulphur and phosphorous as functional groups (Fig. 3).

FTIR spectroscopy allows the qualitative determination of organic compounds as the characteristic vibrational mode of each molecular group causes the appearance of bands in the infrared spectrum at a specific frequency, which is further influenced by the surrounding functional groups[25] FTIR spectra of both samples were taken in the region of 400-4000 cm-1. General overview of all the samples indicates presence of large number of functional groups.

Differentiation between Aśudhdha and Śodhita sample can seen at the range of 1200 to 500 cm-1 s. Six wavelength were selected for matrix plotting. i.e. 1161, 1062, 818, 777, 751, 556 cm-1 for easy discrimination. These frequencies are associated with Alcohol and Phenols, Amine, Carboxylic acid, Sulphur fuctional group and Phosphorus function group. Among them, Śodhita and Aśodhita samples can easily differentiated at λ 1161, λ1062 and λ556 with all combination. Similarity was also found between the wavelength range 1000 to 600 cm-1. Five wavelengths were selected for matrix plotting. i.e.1023, 945, 712, 647, 660 cm-1. samples are found similar at this range which suggests that compounds at this range do not change after Śodhana procedure.

Table 6

Observed frequencies of Aśuddha Guggulu in FTIR study

Observed peakRangeIntensityAssignmentVibrationFunctional group
2928.25 2858.742850-3000strCH3, CH2 & CH 2 or 3 bandsAsymmetric strechAlkanes
1376.46 1455.711350-1470 1370-1390Med MedCH2 & CH3 deformation CH3 deformationSymmetric band

3421.603200-3550StrO-H (H-bonded), usually broadAsymmetric strechAlcohols & Phenols
1376.461330-1430MedO-H bending (in-plane) Symmetric band 

3421.603400-3500 (dil. soln.)WkN-H (1°-amines), 2 bandsAsymmetric strechAmines
1659.221550-1650med-strNH2 scissoring (1° amines) Symmetric band

1711.331710-1720StrC=O (saturated ketone)Asymmetric strechAldehydes & Ketones
1455.711400-1450Stra-CH2 bendingSymmetric band

1659.221630-1695 (amides)StrC=O (amide I band)Asymmetric strechCarboxylic Acids & Derivatives
1517.051500-1560MedN-H (2-amide) II bandSymmetric band

1164.88 1125.111050-1200StrC=S thiocarbonylstrechSulphur Functions
1036.581030-1060StrS=O sulfoxidestrech
1376.46 & 1164.881365 ± 5 & 1180± 10Strsulfonyl chlorideAsymmetric & symestric both strech

1036.58900-1050StrP-OR estersstrechPhosphorous Functions
1125.111100-1200StrP=O phosphine oxide/phosphatestrech
1243.881230-1260 1200-1275StrPhosphonate/Phosphoramidestrech

 1250± 10str & shpSi-CH3StretchSilicon Functions

Str - Strong, Med – Medium, Wk - Weak, Shp - Sharp

Table 7

Observed frequencies of Śuddha Guggulu in FTIR study

Observed peakRangeIntensityassignmentvibrationFunctional group
2861.14
2929.84
2850-3000strongCH3, CH2 & CH
2 or 3 bands
Asymmetric
strech
Alkanes
1385.09
1453.03
1350-1470MedCH2 & CH3
deformation
Symmetric bend
1385.091370-1390MedCH3 deformation

3422.723200-3550StrO-H (H-bonded),
usually broad
Asymmetric
strech
Alcohols & Phenols
1385.091330-1430MedO-H bending (in-
plane)
Symmetric bend

1040.88
1074.42
1040-1100StrO-CAsymmetric
strech
Carboxylic Acids &
Derivatives
1658.901630-1695
(amides)
StrC=O (amide I band)strech

525.75500-540 (wk)WkS-S disulfidestrechSulphur Functions
1040.881030-1060 (str)StrS=O sulfoxidestrech

2333.682280-2440 cm-1med & shpP-H phosphineStretchPhosphorous
1040.88900-1050StrP-OR esters Functions

Str - Strong, Med - Medium, Wk – Weak, Shp - Sharp

Table 8

Results of microbial overload in Aśuddha and Śuddha Guggulu

Microbial growth Samples Permissible Limit (API)
 AG SG-
Total plate count722cfu/g 852cfu/g105cfu/g
Total fungal countAbsent Absent103cfu/g
Escherichia coliAbsent AbsentAbsent
Pseudomonas aeruginuosaAbsent AbsentAbsent
Staphylococcus aureusAbsent AbsentAbsent
Salmonella spp.Absent AbsentAbsent
Table 9

Result of Heavy metal analysis in Aśuddha and Śuddha Guggulu

Heavy metals Samples Permissible Limit (API)
 AG SG-
Lead (Pb)Not Detected Not Detected10ppm
Cadmium (Cd)Not Detected 0.100ppm0.30
    ppm
Arsenic (As)Not Detected 0.606ppm3 ppm
Mercury (Hg)0.575ppm Not Detected1 ppm

ppm= parts per million

Microbial overload and Heavy metal content test

Results of Microbial overload and Heavy metal analysis were placed at Table 8 and 9.

Figure 3

FTIR graph of AG and SG Total

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Total plate counts were within the normal range in all drug samples and total fungal count, Escherichia coli, Psudomonos aeruginosa, Staphylococcus aureus and Salmonella spp were absent. In the heavy metal analysis lead was not detected in any sample. Cadmium was present in minimum amount viz. 0.100 ppm in SG which is within the permissible limits. Arsenic was found 0.606ppm in SG which is also within the permissible limits. Mercury was found 0.575ppm in AG which is nearer to permissible limits. As Guggulu is plant product and collected from arid area, chances of contamination of arsenic, cadmium and mercury may be during storage process. The microbial load and heavy metal analysis authenticated the safety aspect of the formulation from the analytical perspective.

CONCLUSION

Guggulu Śodhana procedure is very important in Āyurvedic pharmaceutics. Guggulu Śodhana was carried out in ten batches of 1 Kg each of Aśuddha Guggulu and 4 l of Gomūtra. Average 7 h was the time duration required for dissolving and evaporation procedure. For sun drying it took 3 days. An average of 80.56% yield was obtained. Average 187.9 g residue was found. Alteration was found in physico-chemical, HPLC and FTIR analysis which show the impact of classical Śodhana procedure on Guggulu. The adopted method for Guggulu Śodhana can be considered as easy, convenient and standard manufacturing procedure. Data obtained from the present study is reproducible. The values of physicochemical parameters can be taken for quality assurance.

GRAPHICAL ABSTRACT

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ACKNOWLEDGEMENT

Authors acknowledge authority of IPGT & RA, Jamnagar for the financial assistance and providing facilities for the study.

ABOUT AUTHORS

Dr.Kuti Yagneshkumar Vyas MD (Ayu) PhD presently serving as Assistant Professor in Sri O H Nazar Ayurveda College, Surat, India obtained her MD and PhD from Gujarat Ayurveda University, Jamnagar in 2015. Contributed to concept, design, literature study, Definition of intellectual content, Pharmaceutical experiments , Data acquisition, Data analysis, Manuscript preparation, Manuscript editing, Manuscript review and Guarantor of this article.

Dr. Vinay Janardan Shukla Msc PhD is currently serving as Head in department of Pharmaceutical Laboratory, IPGT & RA, Gujarat Ayurved University, Jamnagar. He obtained his PhD degree in Analytical chemistry from Sauratra University, Rajkot, Gujarat. He has 30 yrs of research experience. He is Author of 201 research papers, 3 monographs, 1 chapter in book. He is co-investigator in 18 projects of CCRAS and chief investigator in other projects. Contributed to Concept, Design, Pharmaceutical experiments, Data analysis, Manuscript editing and Manuscript review of this article.

Dr. Galib Ruknuddin, MD (Ayu) PhD is currently serving as Associate Professor in department of Rasashastra and Bhaishajya Kalpana at All India Institute of Ayurveda, New Delhi. Previously, he served at IPGT & RA, Gujarat Ayurved University, Jamnagar till October 2016. He has more than 10 Years PG Teaching in concerned subject. Guided more than 20 theses, contributed around 10 chapters and investigated 3 projects as PI / Co-PI. Contributed to Concept, Design, Definition of intellectual content, Data analysis, Manuscript editing and Manuscript review of this article.

Dr. Pradeep Kumar Prajapati, MD (Ayu) PhD is working as Dean, Prof and Head, Department of Rasashastra and Bhaishajya Kalpana, All India Institute of Ayurveda, New Delhi, India. He is a former Director of IPGT & RA, GAU Jamnagar and In - Charge Director of Pharmacy Gujarat Ayurved University Jamnagar, Head, Dept of RS & BK, IPGT& RA, Jamnagar. He obtained Teachers excellence award by CEE, Rasaccharya Award for contributions to Rasashastra, Nagarajuna Silver Medal for MD dissertation, Best Research article Award. He has authored 264 research articles and 3 monographs. Contributed to concept, design, Definition of intellectual content, Data analysis, Manuscript editing and Manuscript review of this article.

Notes

[5] Conflicts of interest CONFLICT OF INTEREST Nil

REFERENCES

1. 

Sharma Ācharya Sadānada, Taraṅgiṇi Rasa , authors. 2nd Taraṇga/52. 11th. New Delhi: Motilāla Banārsidas;; 2004. p. 21

2. 

Suṣruta Ācharya , author. Suṣruta Saṃhitā of Suṃruta. Sūtra sthāna 15/32. Ācharya YT , editor. Varānasi: Chaukhambha Surbhārti Prakāśan; 2003. p. 73

3. 

Nagarajan M, Waszkuc TW, Sun J , authors. Simultaneous determination of E-and Z-guggulsterones in dietary supplements containing Commiphora mukul extract. J Assoc Off Anal Chem Int 2001;84(1):24-8. 2001. 84(1):p. 24–8

4. 

newhope.com [New Hope Network] Boulder, CO 80301:Nutrition Business Journal, Ingredient 5: Ayurveda gains ground in US market on strength of specific herbs Oct-2012.Retrieved date:12/8/2016. Available from: http://www.newhope.com/ingredients-general/ayurveda-gains-ground-us-market-strength-specific-herbs.

5. 

krishna.nic.in [Krishna District Official Website] Plantation and Extraction of Guggul. cited 2016 Nov 23Available from: http://krishna.nic.in/PDFfiles/MSME/Herbal/guggul[1].pdf.

6. 

Masten SA , author. Gum guggul and some of its steroidal constituents: review of toxicological literature. Integrated Laboratory Systems, Inc. Research Triangle Park, North Carolina, USA.: Document prepared for National Toxicology Program (NTP), National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health, U.S Department of Health and Human Services ; 2005. 2(1):p. 1–49

7. 

Sangle VD, Nadkarni SD, Vahalia MK, Darp MS , authors. The study of effect of ayurvedic processing of Commiphora wightii on gastric irritancy index in experimental animals. Indian Drugs. 2004;70(3):368–72

8. 

Kamble Rachana, Sathaye Sadhana, Shah DP , authors. Evaluation of antispasmodic activity of different Śodhit guggul using different Śodhan process. Indian J Pharm Sci. 2008;70(3):368–72

9. 

Vyas KY, Dhruve K, Prajapati PK , authors. Methods of Guggulu Śodhana in Ayurveda – A Review. Int J Ayu Med. 2014;5(2):154–60

10. 

The Ayurvedic Formulary of India, second revised English. Delhi: Controller of publication; 2003

11. 

Randhawa GK , author. Cow urine distillate as bioenhancer. J Ayurveda Integr Med. 2010;1:240–1

12. 

Devender O, Sachdev, Devesh D, Gosavi, Salwe KJ , authors. Evaluation of antidiabetic, antioxidant effect and safety profile of Gomūtra ark in Wistar albino rats. Anc Sci Life. 2012;31(3):84–9

13. 

Jain NK, Gupta VB, Garg R, Silawat N , authors. Efficacy of cow urine therapy on various cancer patients in Mandsaur District, India - A survey. Int J Green Pharmacy. 2010;4(1):29–35

14. 

Pimpale SA, Patil A, Desai S , authors. Comparative analytical study of Guggulu (Commiphora mukul) Śodhana done in different media. Int Ayu Medi J. 2014;2(5):761–4

15. 

Sharma K, Lather A, Kumar V, Tyagi V, Lather NA , authors. Study On Standardization Of Triphalāa Śodhita Guggulu (Commiphora mukul Hook). World J of Pharma Res. 2014;3(2):2156–62

16. 

Hārῑta Ācharya, Saṃhitā Hārῑta , authors. Kalpa Sthāna 5/9. Tripathi Hariprasad , editor. Varanasi: Chaukhamba Chaukhambha Krishnadas Academy; 2009. p. 511

17. 

Chaube Dattaram, Suṅdar Bṛhat Rasarāja , authors. Madhyam khaṅda Shilajῑta prakaraṇa. 3rd. Varanasi: Chaukhamba Orientlia; 2000. p. 185

18. 

Anonymous,The Ayurvedic Pharmacopoeia of India. 1st ed, Vol I, Part-II. New Delhi: Controller of publication, Dept. of I.S.M. and H. Ministry of Health and Family Welfare, Govt. of India; 2007. Appendix 2.

19. 

Gupta AK, Tandon N, Sharma M , authors. Quality standards of Indian Medicinal Plants Vol 3. New Delhi: ICMR; 2005. p. 179

20. 

Sharma S, Ras Sharma S. , authors. Tarṅgiṇῑ 24/579-580. Shastri Haridatta , editor. Delhi: Motilāl Banārasidās Prakāshana; 1986. p. 754

21. 

The Ayurvedic Pharmacopoeia of India, Vol I, Part-I. New Delhi: Controller of publication, Dept of ISM and H, Ministry of Health and Family Welfare, Govt. of India; 2001. p. 43

22. 

Shishodia S, Kuzhuvelil HB, Dass S, Krishan SG, Agarwal BB , authors. The Guggul for Chronic Diseases: Ancient Medicine, Modern Targets. Anticancer Research. 2008;28:3647–64

23. 

Jasuja ND, Choudhary M, Sharama P, Sharma N, Joshi SC , authors. A review on bioactive compounds and medicinal uses of Commiphora mukul. J Plant Sci. 2012;7:113–37

24. 

Kulhari A, Sheorayan A, Chaudhary A, Sarakar S, Kalia R , authors. Quantitative determination of guggulsterone in existing natural populations of Commiphora wightii (Arn.) Bhandari for identification of germplasm having higher guggulsterone content. Physiol Mol Biol Plants . 2015;21(1):71–81

25. 

Grube M, Muter O, Strikauska S, Gavare M, Limane B , authors. Application of FT-IR spectroscopy for control of the medium composition during the biodegradation of nitro aromatic compounds. J Ind Microbiol Biotechnol. 2008;35:1545–9