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Year : 2018 | Volume : 45 | Issue : 2 | Page : 67-71
Sankeshwari RM
Soxhlet versus cold maceration: Which method gives better antimicrobial activity to licorice extract against Ankola AV
Streptococcus mutans? Bhat K
Hullatti K
Roopali M Sankeshwari1, Anil V Ankola1, Kishore Bhat2, Kirankumar Hullatti3
1 Department of Public Health Dentistry, KLE Academy of Higher Education and Research's, KLE VK Institute of Search in Google Scholar
Dental Sciences, Belgaum, Karnataka, India for
2 Dr. Prabhakar Kore Basic Science Research Centre, KLE VK Institute of Dental Sciences, Belgaum, Karnataka, India
Sankeshwari RM
3 Department of Pharmacognosy, KLE Academy of Higher Education and Research's, College of Pharmacy, Belgaum,
Ankola AV
Karnataka, India Bhat K
Hullatti K
Date of Web Publication 10-Dec-2018
Related articles

Cold maceration
licorice root extract
Soxhlet method
Correspondence Address:
Roopali M Sankeshwari
Department of Public Health Dentistry, KLE VK Institute of Dental Sciences, KLE Academy of Higher Education and Access Statistics
Research (KLE University), Belgaum, Karnataka Email Alert *
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In this article
Abstract
Check Introduction
Methodology
Results
DOI: 10.4103/jss.JSS_27_18 Discussion
Conclusion
References
Article Tables
Abstract
Article Access Statistics
Purpose: Licorice is called “grandfather of herbs” and is being used for wide various ailments since time immemorial. Viewed 6232
However, its use in dentistry has been recently. Soxhlet and Cold maceration are the two commonly employed methods Printed 207
for extraction of drug from raw products. But which of two is gives better antibacterial property to licorice root remains Emailed 0
unanswered. Hence, the present study has been planned with an aim to compare antibacterial activity of licorice root PDF Downloaded 597
extracts obtained from two methods (Soxhlet and cold maceration) against Streptococcus mutans. It is an in vitro study.
Comments [Add]
Methodology: Licorice roots were authenticated from recognized taxonomist. They were washed, dried completely, and
coarsely powdered. The weighed powder was mixed with ethanol (100 mg in 500 ml). Two such mixtures were made. Cited by others 1
One was used for cold maceration procedure and the other was used for Soxhlet method. Extracts so obtained were
assessed for their minimum inhibitory concentration against S. mutans ATCC 25175 in triplicates using broth dilution and
disc diffusion method. Extracts were also compared for their phytochemical components. Descriptive analysis and
unpaired t-test were performed. Results: Cold maceration extract at concentration of 1.95 mg/ml and Soxhlet method at
3.906 mg/ml showed inhibition of S. mutans. Both of them possessed the same phytochemical components. Conclusion:
Licorice root extract obtained through cold maceration had significantly better antimicrobial activity against S. mutans
than licorice extract obtained through Soxhlet method. Cold maceration method is relatively simple and does not involve
complex instruments and yet yields better extract.

Keywords: Cold maceration, licorice root extract, Soxhlet method

How to cite this article:

Sankeshwari RM, Ankola AV, Bhat K, Hullatti K. Soxhlet versus cold maceration: Which method gives better
antimicrobial activity to licorice extract against Streptococcus mutans?. J Sci Soc 2018;45:67-71

How to cite this URL:

Sankeshwari RM, Ankola AV, Bhat K, Hullatti K. Soxhlet versus cold maceration: Which method gives better
antimicrobial activity to licorice extract against Streptococcus mutans?. J Sci Soc [serial online] 2018 [cited 2020 Aug
27];45:67-71. Available from: http://www.jscisociety.com/text.asp?2018/45/2/67/247150

Introduction

Licorice (Glycyrrhiza glabra) called as Madhuyashti, Mulethi, and Yastimadhu is an herbaceous perennial plant. Root
consists of stolons and pieces of roots. It occurs in Southern Europe, Spain, Syria, Russia, Egypt, Arab, and Iran.[1] In
India, it is reported to be cultivated in Baramulla, Srinagar, Jammu, Dehradun, Delhi, and South India. Licorice is
obtained from the unpeeled, dried roots and stolons of G. glabra and Glycyrrhiza uralensis. Licorice contains several
classes of secondary metabolites such as coumarin, flavonoids, isoflavonoids, pterocarpenes, saponins, and stilbenes
which have been described in detail by Wang and Kondo.[2] Licorice has been used in Ayurveda since time immemorial.
It is 50 times sweeter than sucrose and is used as diuretic, demulcent, mild laxative, aphrodisiac, expectorant, hemostatic,
and intellect promoting.[3] The active chemical ingredients imparting the unique licorice taste are glycyrrhizic acid and
its glucoside, glycyrrhizin (C42H62O16). These molecules are regarded as nearly synonymous as powerful organoleptic
flavorants and impart characteristic licorice taste and aroma.[4],[5]

Literature has a report of licorice being used for oral diseases such as dental caries, periodontal diseases, candidiasis, and
recurrent aphthous ulcers.[6] It has also been used as a mouthwash and lollipop for control of tooth decay.[7],[8] The
aciduric mutans streptococci (MS) group, including Streptococcus mutans and Streptococcus sobrinus, are highly
cariogenic and represent microorganisms most closely associated with dental caries. The oral cavity contains at least 52
genetic strains of S. mutans.[9],[10] Some MS strains may have enhanced abilities to adhere and propagate in specific oral
environment [11] including selective colonization of hard-tissue sites.[12] In addition, several MS strains have been
known to concurrently colonize a single tooth, and single genotypes have been identified to colonize multiple sites within
the dentition of individual patients.[13] Hence, S. mutans is considered as the main culprit for dental caries and was thus
selected for the present study.

Ayurvedic products are polyherbal by nature and are found in the crude form. There are many extraction procedures
which are available to obtain the desired drug. Most commonly followed extraction methods are Soxhlet method and cold
maceration. Soxhlet extraction has been used for many decades, is very time-consuming, and requires relatively large
quantities of solvents. It also needs Soxhlet extractor special equipment for the process.

On the other hand, cold maceration is a simple procedure which does not require any special armamentarium. Cold
maceration always results in an odor similar to that in the original plant material without causing degradation of the
thermolabile compounds present in the fraction due to the low extraction temperature similar to cold pressing.[14]

Although various methods have been described in the literature for extraction of crude drug, a scientific comparative
study to know which method is superior over the other is lacking for licorice extract. Hence, the present study has been
planned with an aim to compare the antibacterial activity of licorice extract obtained using cold maceration and Soxhlet
method against S. mutans.

Methodology

The present study is an in vitro study, and ethical approval was obtained from the Institutional Ethics Committee No. 820
dated January 28, 2014, KLE University.

For licorice extract preparation, dried licorice roots were procured from KLE Ayurveda Pharmacy, Belgaum, Karnataka.
Materials used in this in vitro study are as follows:

Licorice roots
Pure ethanol
Standard strain of S. mutans ATCC 25175.

Licorice extract was prepared using two different methods which are cold maceration and Soxhlet method.

Cold maceration

Purchased licorice root specimen was authenticated from recognized botanist at Indian Council of Medical Research's
Regional Medical Research Centre, Belgaum. After washing all the roots, they were dried in the shade for 3–4 days.
Roots were cut into small pieces in a grinder and were grounded to coarse powder. One hundred grams of licorice powder
was mixed with 500 ml of 100% ethanol in a conical flask. The mixture was stirred thoroughly with a glass rod. The
conical flask was kept with intermittent shaking for 72 h. The mixture was filtered using muslin cloth and through
Whatman No. 1 filter paper. The filtrate was concentrated using an IKA rotary evaporator at 40°C, and the resultant
residue was kept in a refrigerator till further use.

Soxhlet method

One hundred grams of coarse powder of licorice root was packed in a muslin cloth bag and placed in the body of Soxhlet
extractor. Then, 500 ml of ethanol (solvent) was poured in the round-bottom flask. The apparatus was then fitted with the
help of clamps and stand to support the Soxhlet extractor, round-bottom flask, and condenser. The rubber tube connected
to the tap water was attached to the condenser for continuous flow of water. The solvent was heated using the isomantle,
which began to evaporate, moving through the apparatus to the condenser. The condensate then dripped into the reservoir
containing the plant extract. Once the level of solvent reached the siphon, it poured back into the flask and the cycle
began again. The process was made to run for a total of 6 h. Finally, the extract was collected in the round-bottom flask.
Once the process was finished, the ethanol was evaporated using IKA rotary evaporator at 40°C, leaving a small yield of
extracted plant material (about 2–3 ml) in the glass-bottom flask. Extract was kept in a porcelain bowl till the remaining
ethanol was completely evaporated. The content of extractable matter was calculated in mg/g of air-dried material using
digital weighing balance. The extract was stored in the refrigerator till further use.

Phytochemical screening

The ethanolic extracts obtained from both the techniques were subjected to preliminary phytochemical screening for
qualitative detection of phytoconstituents using standard procedures as described by Trease and Evans.[15]

Preparation of inoculums

The microbial strain used for this study was procured from the Institute of Microbial Technology, Chandigarh (ATCC
25175). Stock cultures were maintained at 4°C on the slant of nutrient agar. Active cultures for experiments were
prepared by transferring a loop full of cells from the stock cultures to test tubes of nutrient broth for bacteria which were
incubated for 24 h at 37°C.

Determination of antimicrobial activity of the licorice extract was done as per the Clinical Laboratory and Standard
Institute guidelines.[16]

The lowest concentration of the extracts that inhibits the growth of test organisms is the minimum inhibitory
concentration (MIC). The MIC of the licorice extracts was determined by broth dilution method against bacterial
culture
MIC of the extract against S. mutans (ATCC 25175) by broth dilution method was carried. Media used was brain–
heart infusion (BHI) broth. Culture/inoculum: S. mutans. Stock solution of the extract: 50% (500 mg in 1 ml of
dimethyl sulfoxide).
Extracts: Licorice extract-Cold maceration method (LC)

Licorice extract-Soxhlet extraction method (LS).

Procedure

Nine dilutions of extract were done with BHI for MIC. In the initial tube, only 200 μl of extract was added. For dilutions,
200 μl of BHI broth was added into the next nine tubes separately. In the 2nd tube, 200 μl of extract was added which
already contains 200 μl of BHI broth. This was considered as 10−1 dilution. From 10−1 diluted tube, 200 μl was
transferred to the second tube to make 10−2 dilution. The serial dilution was repeated up to 10−8 dilution for each extract.
From the maintained stock cultures of required organisms, 5 μl was taken and added into 2 ml of BHI broth. In each
serially diluted tube, 200 μl of above culture suspension was added. The last tube contained only the media and culture
suspension. The tubes were kept for incubation for 24 h at 37°C in bacteriological incubator and observed for turbidity.
The experiment was repeated in triplicate for both LS and LC, to ascertain the antimicrobial activity.

Disc diffusion method was carried out to confirm the results of broth dilution method.

Results

Approximately 8 g and 6.5 g of licorice extract were obtained from 100 g of powder through cold maceration and Soxhlet
method, respectively.

For broth dilution method of MIC, turbidity was seen in the ninth tube of cold maceration method and eight tube of
Soxhlet method.

Further result was confirmed with the help of disc diffusion method in which the zone of inhibition was noted. All the
experiments were repeated in triplicates, and the average value was taken.

[Table 1] shows the MIC values of the triplicate experiments. The mean MIC value for cold maceration was 1.8 ± 0.145,
and for Soxhlet method, it was 3.8 ± 0.097 [Table 2]. When unpaired t-test was applied, P = 0.000 with 95% confidence
interval which concluded that there was a statistically significant difference between MIC values obtained through two
methods [Table 3]. When phytochemical screening was performed, both the extracts showed the presence of components
as shown in [Table 4].
 Table 1: Minimum inhibitory concentration values of the cold maceration and Soxhlet
method

Click here to view

Table 2: Mean and standard deviation of the minimum inhibitory concentration value

Click here to view

Table 3: Comparison of antimicrobial activity of licorice extract obtained by cold
maceration and Soxhlet method (independent t-test)

Click here to view

Table 4: Phytochemical test results of both the extracts

Click here to view

Discussion

Licorice has been used in Ayurveda for many ailments, and the literature search shows that it has good scope to be used
in medicine and dentistry as an adjunct to allopathic drugs. Its effectiveness on general health problems has been well
documented, but its effect on S. mutans, the main culprit in the initiation of dental caries, is sparsely reported.[17]

Ayurvedic drug formulation is known as pancavidhaa kasayaa and has description of five basic forms: Swarasa, Kalka,
Kwatha, Sheeta, and Faanta. Ayurveda believes that a plant as a whole may not have therapeutic effect, and hence, the
active ingredients need to be extracted from the whole plant.[18] The type of extraction procedure selected should be
based on the nature of the constituents. If the constituents are thermolabile, then extraction procedures such as cold
maceration and percolation are preferred.[18] The major constituents of licorice are triterpenoids and flavonoids, apart
from small quantities of pyrrolopyrimidine alkaloid and tetrahydroquinoline alkaloids.[19],[20],[21] The chemical
components responsible for antioxidant and antibacterial activity present in G. glabra roots have been reported such as
glycyrrhizin, glycyrrhizinic acid, glabridin, glabrene, glabrol, licoflavonol, glycyrol, licoricone, formononetin,
phaseollinisoflavan, hispaglabridin A and B, 3-hydroxyglabrol, and 3 methoxyglabridin, glabranin isomer.[22],[23],[24]
Flavonoids and phenylpropanoids can degrade when kept in organic solvents. Glycosides tend to break up when exposed
to higher temperatures as in case of Soxhlet extraction. We can contemplate that differences in MIC values observed in
the present study could be attributed to differences in flavonoid concentration which is responsible for antibacterial
activity of licorice to a large extent.

Solvent used for extract of crude product also plays an important role. Water and alcohol are the two commonly used
solvents for extraction procedure. Water is considered as a universal solvent as it is inert, but it may not be useful in all
circumstances, especially when the ingredients are insoluble in water. Hence, alcohol is considered a good choice for
such conditions, and moreover, it has many advantages over water such as:

Alcohol is neutral and hence extract products obtained from it are compatible with other products, small amount of
heat is required to concentrate the alcoholic preparations, and it dissolves selective active constituents of the drug.
[23] This postulate was tested by Ahmad et al.[24] and Jain et al.[8] who concluded that ethanolic extract of licorice
had better antimicrobial activity. This might be attributed to the polar nature of the solvent ethanol which resulted
in leaching of more active ingredients during extraction.

The phytochemical screening revealed the presence of carbohydrates, reducing sugars, terpenoids, glycosides, steroids,
tannins, saponins, anthraquinones, flavonoids, and alkaloids. The presence of these secondary metabolites could
contribute to the antibacterial activity of the extracts. It is interesting to note that these components were present in both
the extracts, but however, the MIC value of both differed significantly. This difference could be attributed to the
difference in solubility of both the extracts. In the present study, the cold maceration extract dissolved completely than
extract obtained by Soxhlet method.

A comparison between different methods for extraction of glycyrrhetic acid from licorice stolons was reported by Sharad
Visht [25] which concluded that extraction ratio of glycyrrhetic acid can be increased by changing pH of extraction
solvent. In the present study, cold maceration proved to be better than Soxhlet method for extraction of licorice roots. The
study result is of practical significance which demonstrates that licorice extract obtained from simple method of cold
maceration is superior to the extract obtained from Soxhlet method which needs special apparatus for carrying out the
procedure along with continuous supply of tap water for at least 24 h apart from requirement of personnel to monitor the
procedure. Hence, future studies which are planned on licorice roots can directly adopt cold maceration method for
obtaining extract.

The study has some limitations such as the use of single organism. It is a known fact that dental caries is a multifactorial
disease and etiology has been attributed to various causative microorganisms. However, S. mutans is predominant of all
cariogenic microorganisms, and hence, it was selected in the present study. In future, studies can be conducted on various
other cariogenic microorganisms to know if licorice extract has broad-spectrum antimicrobial activity. Confirmatory tests
such as thin-layer chromatography (TLC)/high-performance TLC can be carried out to know if there is any difference in
the concentration of active ingredients of both cold maceration and Soxhlet extract.

Conclusion

The present study concludes that the licorice root extract obtained from cold maceration method showed significantly
better antimicrobial activity than extract obtained from Soxhlet method against S. mutans.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1. Gyanendra Pandey. Drvyaguna Vigyan. Vol II. New Delhi: Chavkhamba Publication; 2004. p. 456-68.

2. Tao R, Tong Z, Lin Y, Xue Y, Wang W, Kuang R, et al. Antimicrobial and antibiofilm activity of pleurocidin
against cariogenic microorganisms. Peptides 2011;32:1748-54.

3. Sastry DJ. Dravyagunn Vijnana. Varanasi: Chavkhamba Publication; 2013. p. 152-6.

4. Touyz LZ. Liquorice health check, oro-dental implications, and a case report. Case Rep Med 2009;2009:170735.

5. de Marsillac Mde W, Vieira Rde S. Assesment of artificial caries lesions through scanning electron microscopy and
cross-sectional microhardness test. Indian J Dent Res 2013;24:249-54.
6. Messier C, Epifano F, Genovese S, Grenier D. Licorice and its potential beneficial effects in common oro-dental
diseases. Oral Dis 2012;18:32-9.

7. Peters MC, Tallman JA, Braun TM, Jacobson JJ. Clinical reduction of S. Mutans in pre-school children using a
novel liquorice root extract lollipop: A pilot study. Eur Arch Paediatr Dent 2010;11:274-8.

8. Jain E, Pandey RK, Khanna R. Liquorice root extracts as potent cariostatic agents in pediatric practice. J Indian
Soc Pedod Prev Dent 2013;31:146-52.
[PUBMED] [Full text]
9. Napimoga MH, Höfling JF, Klein MI, Kamiya RU, Gonçalves RB. Tansmission, diversity and virulence factors of
Sreptococcus mutans genotypes. J Oral Sci 2005;47:59-64.

10. Tabchoury CP, Sousa MC, Arthur RA, Mattos-Graner RO, Del Bel Cury AA, Cury JA, et al. Evaluation of
genotypic diversity of Streptococcus mutans using distinct arbitrary primers. J Appl Oral Sci 2008;16:403-7.

11. Svensäter G, Larsson UB, Greif EC, Cvitkovitch DG, Hamilton IR. Acid tolerance response and survival by oral
bacteria. Oral Microbiol Immunol 1997;12:266-73.

12. Baca P, Castillo AM, Baca AP, Liébana MJ, Junco P, Liébana J, et al. Genotypes of Streptococcus mutans in saliva
versus dental plaque. Arch Oral Biol 2008;53:751-4.

13. Hardie JM. Oral Streptococci. In: Sneath PH, Mair NS, Sharpe ME, editors. Bergey's Manual of Systematic
Bacteriology. Baltimore, MD: Williams and Wilkins; 1986. p. 1054-63, 12.

14. Wu C, Wang F, Liu J, Zou Y, Chen X. A comparison of volatile fractions obtained from Lonicera macranthoides
via different extraction processes: Ultrasound, microwave, soxhlet extraction, hydrodistillation, and cold
maceration. Integr Med Res 2015;4:171-7.

15. Trease GE, Evans W. Pharmacognosy. 14th ed. London, UK: WB Saunder Company Ltd; 1996. p. 612.

16. Cockerill FR, Wikler MA, Alder J, Dudley MN, Eliopoulos GM, Ferraro MJ, et al. Performance standards for
antimicrobial disk susceptibility tests; approved standard. Wayne, Pennsylvania, USA: Clinical and Laboratory
Standards Institute; 2012. p. 76.

17. Sedighinia F, Safipour Afshar A, Soleimanpour S, Zarif R, Asili J, Ghazvini K, et al. Antibacterial activity of
Glycyrrhiza glabra against oral pathogens: An in vitro study. Avicenna J Phytomed 2012;2:118-24.

18. Savrikar SS, Ravishankar B. Bhaishajya kalpanaa - the ayurvedic pharmaceutics - an overview. Afr J Tradit
Complement Altern Med 2010;7:174-84.

19. Rao KV. A review on licorice. Anc Sci Life 1993;13:57-88.

20. Fukai T, Tantai L, Nomura T. Isoprenoid substitute flavonoid from Glycyrrhiza glabra. Phytochemistry
1996;43:531-53.

21. Gupta VK, Fatima A, Faridi U, Negi AS, Shanker K, Kumar JK, et al. Antimicrobial potential of Glycyrrhiza
glabra roots. J Ethnopharmacol 2008;116:377-80.

22. Gupta A, Maheshwari DK, Khandelwal G. Antibacterial activity of Glycyrrhiza glabra roots against certain gram-
positive and gram-negative bacterial strains. J Appl Nat Sci 2013;5:459-64.

23. Aulton M. Aultons Pharmaceutics :The design and manufacture of medicine. 4th ed. London: Churchill
Livingstone; 2013.

24. Ahmad I, Mehmood Z, Mohammad F. Screening of some indian medicinal plants for their antimicrobial properties.
J Ethnopharmacol 1998;62:183-93.

25. Sharad Visht GK. A comparison between different methods for extraction of glycyrrhetic acid from licorice
stolons. Int J Pharma Prof Res 2012;3:622-6.

Tables

[Table 1], [Table 2], [Table 3], [Table 4]

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