INTRODUCTION:

Guggulu is an oleo gum resin from the plant, Commiphora wightii (Arn.) Bhand., is a highly renowned drug with wide range of pharmacological actions. Guggulu is a main ingredient in various kinds of polyherbal formulations like kashaya (decoction), thaila (oil), Gritha (ghee), Gutika (tablets) and choorna (powder). Important Ayurvedic polyherbal formulations which include Guggulu are Guggulu tiktakam kashaya, Guggulu marichadi thaila, Guggulu tiktakam ghritha, Kaisora guggulu gutika, Guggulu panchapala choorna etc.

Ayurveda recommends gugguluin various disease conditions, including medoroga (hyperlipidaemia), vata roga (arthritic conditions), gandamala (thyroid disorders), hridroga (heart diseases), kushta (skin disorders), vrana (wounds)¹ etc. The main active phytoconstituents identified in Guggulu are Guggulsterone E & Z. They are responsible for the wide range of pharmacological actions such as anti-inflammatory, anti-arthritic, anti-hyperlipidemic, anti-oxidant, thyroid stimulatory, anti-microbial, cardioprotective, anti-diabetic, anti-angiogenesis etc². Even though both Guggulsterones had actions such as anti-angiogenesis³, thyroid stimulatory⁴ and wound healing⁵, Guggulsterone Z had a greater effect on these. Thus, in quality control they are considered as biomarkers.

There are several methods of Shodhana (purification) practiced in Ayurveda including soaking, grinding, boiling and roasting in particular media. These methods are designed to remove physical as well as chemical impurities, to reduce the adverse effects and to enhance the efficacy. It is advised that Guggulu should be purified before being added to formulations. However, in Ayurvedic texts, there is no mention of adverse effects for Guggulu. Various clinical studies have reported adverse effects associated with intake of crude Guggulu including gastric irritation, skin rashes, mild nausea, headache and with very high doses liver toxicity⁶.

Many Shodhana (purification) processes in different liquid media are described for Guggulu in various Ayurvedic classical texts. The type of Shodhana (purification) mentioned for Guggulu is boiling. Various studies are available regarding the purification of Guggulu in different liquid media such as gomutra (Cow’s urine), godugdha (milk), triphala kwatha (decoction of fruit rinds of Terminalia chebula, Terminalia belleirica and Phyllanthus emblica), vasa patra swarasa (leaf juice of Adhatoda vasica), Vasa patra kwatha (decoction of leaf of Adhatoda vasica)^7,8 etc. In those studies, probable effect on the quantity of Guggulsterones E & Z before and after shodhana of Guggulu were evaluated.

There is no study reported on the shodhana of Guggulu in tila thaila (sesame oil). Shodhana of Guggulu in tila thaila (sesame oil) is mentioned in Anandakanda, a classical Ayurvedic text, using an apparatus called Dolayantra (swinging apparatus - specially designed apparatus used for the purpose of boiling)⁹. Hence, in the present study, shodhana of Guggulu in sesame oil was accomplished with the aid of Dolayantra (Swinging apparatus) and evaluated the physicochemical variations, quantification of Guggulsterones E and Z using HPTLC as well as GC-MS analysis of Guggulu before and after Shodhana.

MATERIALS AND METHODS:

Collection and identification of plant materials:

Genuine oleo gum resin of Guggulu was collected from its natural habitat (Near Rajasthan, India) and was authenticated in the Pharmacognosy unit, Government Ayurveda College, Thiruvananthapuram, Kerala, India) and voucher specimen (No. DGAVC 202/19) was deposited in the Herbarium Unit of Department of Dravyaguna vijnana, Government Ayurveda College, Thiruvananthapuram, Kerala, India. The study was approved by the ethics committee of Government Ayurveda College, Thiruvananthapuram, Kerala, India with reference number- AVC IEC 330/2018.

Chemicals and reagents:

All reagents used were of HPLC grade and procured from Merck life science, Mumbai, India. Standards (Guggulsterone E and Z) were obtained from Sigma Aldrich, USA.

Procedure of shodhana (purification) of Guggulu:

Crude Guggulu (125 g) was taken and manually removed the foreign matters like stone, dried twigs etc. which got adhered to it. Then this was pounded into small pieces and placed over a piece of cotton cloth which was then tied to loosely wrap the Guggulu to form a pottali (a bundle tied with cloth). An earthen pot (capacity-2L) was taken and half of the pot was filled with 1 litres of tila thaila (sesame oil). A wooden stick was placed over the mouth of the pot and the pottali was tied in this wooden stick and suspended inside the pot so as to immerse the pottali in sesame oil and in such a way that it won’t touch the bottom of the pot (This equipment is called Dolayantra). This Dolayantra was kept on mild fire in a burner. It was boiled continuously, till thaila paka lakshana (appearance of froth around the pottali in sesame oil) attained, after the disappearance of froth during the initial stage of boiling. Here, around 20 minutes taken for attaining thaila paka lakshana. Then the pottali was taken out and the contents were transferred to a stainless steel tray. 130 g of Guggulu was obtained after purification with variation in colour, odour and taste from crude Guggulu. The samples were labelled as cG (crude Guggulu) and pG (purified Guggulu) and stored in air tight glass containers. Fig. 1 shows the raw materials and procedure of shodhana (purification).

Organoleptic Evaluation:

Organoleptic characters like form, colour, odour, taste and surface conditions of crude and purified samples of Guggulu were carefully evaluated using sensory organs¹⁰.

Preliminary Physicochemical and phytochemical Evaluation:

Preliminary Physicochemical analysis and phytochemical screening were conducted as per standard procedures mentioned in Ayurveda pharmacopeia of India (API)¹¹.

HPTLC method for estimation of Guggulsterone E & Z:

Preparation of standard solutions and samples:

Guggulsterone E and Z stock solutions were created separately by dissolving 5mg of each compound in 100 ml of methanol (0.05mg/ml). Separately weighed portions of 2grams of crude and purified Guggulu were then refluxed with methanol for 20 minutes. The volume was then reduced to 10ml and filtered using Whatmann filter paper No. 1.

HPTLC method of quantification and method validation:

Chromatographic conditions: HPTLC was performed on 20cm × 20cm pre-coated silica gel aluminium 60F₂₅₄plates (Merck, Mumbai, India). The solutions were applied using a CAMAG Linomat 5 applicator (Hamilton, Switzerland) as 6mm wide bands. The application volume of sample was 6µl and Standards were 3µl, 5µl, 7µl, 9µl, 11µl and 13µl. The applied plates was kept in twin trough chamber with mobile phase- Petroleum ether: Ethyl acetate (7:2v/v). Densitometric scanning was performed using CAMAG TLC Scanner 3(CAMAG, Switzerland), equipped with WIN CATS software version 1.3.4. at 251nm for Guggulsterone E(GE) and 256nm for Guggulsterone Z (GZ). To get an appropriate regression line having a desirable standard deviation (<3) and regression coefficient (<1), % deviation was fixed, while including maximum sample spots in the regression line. Percentage weight by weight (%w/w) of Guggulsterone E and Z were calculated in relation to the %weight of samples.

Method validation:

The method was validated for the analysis of GE and GZ in the methanol extracts of crude and purified Guggulu. Linearity, specificity, precision, accuracy, LOD and LOQ were examined as per ICH 1996/2005.

GC-MS analysis:

The analysis was carried out using a GC-MS apparatus (Model; TQ 8040 series, Shimadzu). 0.1mg of crude and purified Guggulu were dissolved in 10ml methanol separately and stirred continuously with a glass rod for 20minutes. Through Whatmann no.1 filter paper, the undissolved portion was filtered off and the clear solution was used for analysis. Column: Rxi-5Sil MS (30m × 0.25mm I.D., 0.25µm)

Procedure:

1µL of sample was injected to the injection port of GC. The oven program that was selected had an initial temperature of 60°C for 2min, which then increased to 200°C for 2min at the rate of 5°C/min, which further increased to 200°C for 2 min at the rate of 50°C per minute, which then increased to 220°C for 1min at the rate of 3°C/min. Temperature was increased finally to 250°C at the rate of 6°C/min for 7min. Total run time was 55minutes. The carrier gas used was Helium with purity of 99.999% at a flow rate of 1mL/min. The detector temperature and the injection temperature were 250°C. The sample was injected by split less mode. The ion energy of 70 eV was used for the electron impact ionization. The essential chemical constituents were identified based on their retention time and area percentage using the reference compounds in mass library of NIST and WILEY.

Statistical analysis:

With the help of SPSS Software (PASW statistics 18.0.0), the mean quantities of GE and GZ in Guggulu before and after purification were tested statistically using Paired t test.

RESULTS:

Organoleptic Evaluation:

Organoleptic features of crude and purified Guggulu has been evaluated and given in Table1.

Table 1. Organoleptic characters of crude Guggulu (cG) and purified Guggulu (pG)

	Size and Form	Colour	Surface condition	Odour	Taste
cG	Agglomerated, varying sizes	Yellowish/reddish brown	Rough waxy	Aromatic	Bitter, Astringent
pG	Small, Irregular, agglomerated	Yellowish brown	Rough, crispy, oily	Smell of sesame oil	Bitter

Preliminary Physicochemical and phytochemical evaluation:

Table 2 presents the findings of the physicochemical parameters and phytochemical screening, respectively.

Table 2. Results of physicochemical evaluation and qualitative phytochemical screening of crude Guggulu(cG) and purified Guggulu (pG)

Physicochemical parameters (%)	API Standards	cG	pG
Foreign matter	Not >4	1.27	0.68
Loss of drying	-	4.2	1.4
Moisture content	-	13.03	3.27
Volatile oil	Not < 1	2.8	-
Total ash	Not >5	3.9	6.8
Acid insoluble ash	Not >1	0.8	0.8
Water soluble extractive (cold)	Not <53	55.8	24.41
Alcohol soluble extractive	Not <27	50.07	44.6
Total sugar	-	19.76	10.46
Reducing sugar	-	2.98	8.3
pH (at 36^oC)	-	5.52	5.13
Phytoconstituents	Test
Tannin	Lead acetate test	-	-
Steroids	Salkowaski test	+	++
Phenols	Ferric chloride test	-	+
Alkaloids	Dragendorff’s test	++	++
Saponins	Foam test	-	-
Flavonoids	Shinoda test	+	+

Quantification of Guggulsterones E & Z by HPTLC:

Table 3 represents the results of validation methods of HPTLC for estimation of Guggulsterone E (GE) and Guggulsterone Z (GZ). A linear calibration curve was obtained both via peak area and peak height. The statistical analysis of mean concentrations and graphical representation of concentrations of GE and GZ are shown in Table 4 and Figure. 2 respectively. Fig. 3 gives the Calibration plot via peak area standards.

Table 3. Results of validation studies of HPTLC method for estimation of standards

Validation parameters	GE	GZ
Mobile phase	Petroleum ether: Ethyl acetate (7:2)
Linearity range (ng/spot)	150-550 ng	150-650 ng
Detection wavelength	251 nm	256 nm
Rf value	0.3766±0.0206	0.811±0.0222
Slope mean±SD	10.575±2.72	12.256±2.74
Regression coefficient (r²) ±SD	0.997441±2.72	0.99662±2.74
Limit of detection	0.8487µg	0.7377µg
Limit of quantitation	2.5721µg	2.2356µg
Linearity	Linear	Linear
Specificity	Specific	Specific
Instrumental precision	Precise	Precise
Accuracy	Accurate	Accurate

Table 4. Statistical analysis of mean quantities of GE & GZ in crude Guggulu (cG) and purified Guggulu (pG)

Sample		%w/w of GE (Mean±SD)	%w/w of GZ (Mean±SD)
cG		13.5941±0.0004	44.3698±0.0002
pG		30.8902±0.0003	45.7603±0.0004
Paired Differences
cG-pG	t	299577.240	12042.083
	df	2	2
	Sig. (2-tailed)	0.000	0.000

Figure 2. Mean quantities of GE & GZ in crude Guggulu (cG) and purified Guggulu (pG)

GC-MS analysis:

GC-MS analysis of methanolic extract of crude and purified Guggulu revealed various compounds. GC-MS chromatogram of crude and purified Guggulu are also shown in Figure 4. 19 and 15 compounds respectively of purified and crude Guggulu with their molecular name, peak area percentage and retention time are listed in Table 5.

Figure 4. GC-MS Chromatogram; A –crude Guggulu, B- Purified Guggulu

Table 5. Phytocomponents identified using GC-MS in purified guggulu (pG) andcrude guggulu(cG)

	Name	Area %	Retention time
PURIFIED Guggulu
1	Hexadecanoic acid, methyl ester	7.41	30.194
2	2,4-Diisopropenyl-1-Methyl-1-Vinylcyclohexane	3.64	30.892
3	2,7,11-Cyclotetradecatrien-1-ol, 1,7,11-trimethyl-4-(1-methylethyl)-, [1R(1R,2E,4S,7E,11E)]-	1.51	33.023
4	Methyl 16-R/S-hydroxy-cleroda-3,13(14)-Z-dien-15,16-olide	1.52	33.240
5	9,12-Octadecadienoic acid (Z,Z)-, methyl ester	25.22	34.237
6	9-Octadecenoic acid (Z)-, methyl ester	24.34	34.430
7	Methyl stearate	3.65	35.084
8	6-epi-shyobunol	8.56	35.863
9	4,8,13-Cyclotetradecatriene-1,3-diol, 1,5,9-trimethyl-12-(1-methylethyl)-	1.47	37.399
10	(+)-Longicamphenylone	1.02	37.669
11	12-hydroxy-, 9-Octadecenoic acid(Z)-, methyl ester,	2.34	39.327
12	2-methylhexacosane	1.31	42.190
13	Dotriacontane	1.40	44.237
14	Androstan-3-ol, 9-methyl-, acetate, (3.beta.,5.alpha.)-	2.27	44.745
15	1,4-Methanoazulene, decahydro-4,8,8-trimethyl-9-methylene-, (1S,3aR,4S,8aS)-(+)- (Longifolene)	3.02	44.953
16	Tetracontane	1.49	46.144
17	n-Propyl 9,12-octadecadienoate	1.84	48.048
18	9-Octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester	3.79	48.169
19	Hexatriacontane	1.56	48.426
Crude Guggulu
1	Hexadecanoic acid, methyl ester	1.92	30.172
2	2,4-Diisopropenyl-1-Methyl-1-Vinylcyclohexane	15.00	30.900
3	2,7,11-Cyclotetradecatrien-1-ol, 1,7,11-trimethyl-4-(1-methylethyl)-, [1R(1R,2E,4S,7E,11E)]-	10.40	33.031
4	Thunbergol	6.49	33.230
5	9,12-Octadecadienoic acid (Z,Z)-, methyl ester	1.46	34.169
6	9-Octadecenoic acid(E)-, methyl ester	2.91	34.36 2
7	Cycloheptane, 4-methylene-1-methyl-2-(2-methyl-1-propen-1-yl)-1-vinyl-	1.18	35.378
8	4,8,13-Cyclotetradecatriene-1,3-diol, 1,5,9-trimethyl-12-(1-methylethyl)-	45.99	35.914
9	2,7,11-Cyclotetradecatrien-1-ol, 1,7,11-trimethyl-4-(1-methylethyl)-, [1R(1R,2E,4S,7E,11E)]-	1.60	36.868
10	4,8,13-Cyclotetradecatriene-1,3-diol, 1,5,9-trimethyl-12-(1-methylethyl)-	2.08	37.384
11	Humulane-1,6-dien-3-ol	1.76	37.532
12	(+)-Longicamphenylone	1.55	37.651
13	4,8,13-Cyclotetradecatriene-1,3-diol, 1,5,9-trimethyl-12-(1-methylethyl)-	3.17	40.753
14	1-Bromotriacontane	1.37	42.171
15	Androstan-3-ol, 9-methyl-, acetate, (3.beta.,5.alpha.)-	3.12	44.732

DISCUSSION:

On Organoleptic evaluation, it was observed that purified Guggulu was crispy in nature with the smell of sesame oil. Guggulu that has been purified has a significantly lower pH value than crude Guggulu. It suggests the nature is acidic and might help with antioxidant activity. In the purified Guggulu, foreign matter, loss from drying, and moisture content were decreased, proving its purity. It was noticed that reducing sugar content has been increased in Guggulu after purification. Reducing sugars includes all monosaccharides which had immense anti-inflammatory and cardio protective properties¹². Along with steroid, alkaloid, and flavonoid, purified Guggulu contain phenols. Flavonoids and other Phenolic compounds possess anti-oxidant and as scavengers of free radicals¹³. Steroids helps in anti inflammatory, anti bacterial, antiviral, analgesic and anti oxidant activities¹⁴. Alkaloids have proved to be having properties such as anti microbial, hypotensive, analgesics and anticonvulsant activities¹⁵. Saponins have anti inflammatory activity by inhibition of Cyclooxygenase-2 (COX2)¹⁶. Hence, purified guggulu may possess all these pharmacological actions.

Estimation of Guggulsterones E & Z before and after purification were performed using HPTLC. Guggulsterone E and Z levels increased after purification, with a statistically significant rise (p<0.01). So, after being purified, pharmacological action of Guggulu might have been improved. On GC-MS analysis, area percentage of some compounds like Hexadecanoic acid, methyl ester (rG – 1.92%, pG -7.41%) and 9,12-Octadecadienoic acid (Z, Z)-, methyl ester (rG – 1.46%, pG -25.22%) were found to be increased after shodhana (purification). It has been proved by various studies that 9,12-Octadecadienoic acid (Z,Z)-, methyl ester possess Anti inflammatory, Anti microbial, cancer preventive, hypocholestremic and dermatitigenic activities¹⁷. Hexadecanoic acid, Methyl esterhave anti inflammatory, anti oxidant, hypocholestremic, anti arthritic and analgesic actions¹⁸. 9-Octadecenoic acid (Z)-methyl esterin crude guggulu, which possess anti bacterial and fragrance activity¹⁹, converted to 9-Octadecenoic acid (E)-methyl ester after purification. This compound possess anti microbial, anti inflammatory and hypocholestremic activities²⁰. Newly added phytoconstituents in the purified Guggulu along with the pharmacological actions available from various research papers has been given in Table 6.

Table 6. Pharmacological actions of newly added constituents in purified guggulu

	Phytoconstituents	Activities
1	Methyl stearate	Anti bacterial, anti oxidant²¹
2	6-epi-shyobunol	Anti inflammatory, Anti nociceptive, Antipyretic²²
3	Hexatriacontane	Anti inflammatory, analgesic, Antioxidant²³
4	Dotriacontane	Antimicrobial, antioxidant, antispasmodic²⁴
5	Tetracontane	Anti inflammatory, analgesic²⁵
6	2-methylhexacosane	Antimicrobial, Hypocholestremic²⁶
7	Methyl 16-R/S-hydroxy-cleroda-3,13(14)-Z-dien-15,16-olide	Antimicrobial²⁷
8	12-hydroxy-, 9-Octadecenoic acid(Z)-, methyl ester	Anti viral²⁸
9	9-Octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester	Antimicrobial²⁹
10	1,4-Methanoazulene, decahydro-4,8,8-trimethyl-9 methylene-, (1S,3aR,4S,8aS)-(+)- (Longifolene)	Antioxidant³⁰

CONCLUSION:

Guggulu had distinct variations in its organoleptic characteristics before and after purification. Preliminary physicochemical parameters of the crude and purified samples of Guggulu showed notable variations in terms of moisture content, loss on drying and reducing sugar values. After being purified, the quantity of Guggulsterone E and Z in the Guggulu significantly increased (at p<0.01). The findings of the GC-MS analysis show that after purification, area percentage of some phytocomponents has been increased. Additionally, a few substances were found new to the purified Guggulu. The crude Guggulu is gummy in nature and makes it difficult in adding to the formulations. After purification, the sticky nature of crude Guggulu has been changed to crispy. This is very beneficial in the pharmaceutical industry in the preparation of various types of ayurvedic poly herbal formulations like choorna (powder) and gutika (tablet). The newly added compounds and increased guggulsterones in purified Guggulu could play a role in improved pharmacological actions. It can be inferred that the Ayurvedic purification method, Shodhana (purification), can affect the physical and phytochemical profile and is therefore helpful in enhancing the efficacy of guggulu. Purification also helps to increase the shelf life. Hence, Guggulu purified in sesame oil has applications in both pharmaceutics as well as clinical practice. Further, there is a scope to identify, isolate, and characterise the additional active principles in purified Guggulu for the treatment of variety of diseases.

Abbreviations:

HPTLC - High performance thin layer chromatography

GC-MS - Gas chromatography-mass spectrometry

SPSS - Statistical Package for Social sciences

NIST - National institute of standards and technology

SD - Standard deviation

µl- Micro litre

nm - nanometre

µg - microgram

eV - electron volt

df - degrees of freedom

Sig - significance

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS:

The authors extend their sincere gratitude to all the faculties of Department of Dravyaguna Vijnana and Drug standardisation Unit, Government Ayurveda College, Thiruvananthapuram, Kerala, India to conduct the research.

Funding:

Received funding from Kerala state council for science, technology and environment (KSCSTE) under student project scheme (P Order No 512 Student project 2021).

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Received on 27.07.2023 Modified on 20.11.2023

Research J. Pharm. and Tech 2024; 17(8):3669-3675.

DOI: 10.52711/0974-360X.2024.00572

Size and Form

Colour

Surface condition

Odour

Taste

cG

Agglomerated, varying sizes

Yellowish/reddish brown

Rough waxy

Aromatic

Bitter, Astringent

pG

Small, Irregular, agglomerated

Yellowish brown

Rough, crispy, oily

Smell of sesame oil

Bitter

API Standards

cG

pG

Foreign matter

Not >4

1.27

0.68

Loss of drying

-

4.2

1.4

Moisture content

-

13.03

3.27

Volatile oil

Not < 1

2.8

-

Total ash

Not >5

3.9

6.8

Acid insoluble ash

Not >1

0.8

0.8

Water soluble extractive (cold)

Not <53

55.8

24.41

Alcohol soluble extractive

Not <27

50.07

44.6

Total sugar

-

19.76

10.46

Reducing sugar

-

2.98

8.3

pH (at 36oC)

-

5.52

5.13

Phytoconstituents

Test

Tannin

Lead acetate test

-

-

Steroids

Salkowaski test

+

++

Phenols

Ferric chloride test

-

+

Alkaloids

Dragendorff’s test

pH (at 36^oC)

2,7,11-Cyclotetradecatrien-1-ol, 1,7,11-trimethyl-4-(1-methylethyl)-, [1R(1R,2E,4S,7E,11E)]-