Background

Our prehistoric ancestors sought different natural compounds to cure various ills and diseases so as to improve and enrich their lives, most of these compounds were derived from either plants or animals. Research on traditional medicines has been vital in search for organic pharmaceutical compounds. Animal-based medicines have also been extracted and isolated from different parts of the animal body, from their products (secretions and excrements) or from materials they produce like cocoons and compost in earthworm. A total of 252 essential compounds have been selected by World Health Organization (WHO), of which only 8.7% are from animal origin used as medicine (Costa-Neto, 2005). For almost 4000 years, China has a history of research into medicinal uses of earthworm.

Earthworms are well-known invertebrates, are used extensively as folk fare medicines and claim their successful use in treatment of cancer, ulcer, inflammation, diarrhea, fever, dysentery, tooth ache, etc. (Edwards & Bohlen, 1996). Various studies have shown that earthworms exhibit anti-pyretic, anti-septic, detoxifying, diuretic, anti-hypertensive, anti-allergic, anti-asthmatic, spermatocidal, anti-oxidative, anti-microbial, anti-cancer, anti-ulcer and anti-inflammatory activity (Cooper & Roch, 1984; Cooper et al., 2004; Hori et al., 1974). High protein content of earthworm makes it a desired supplementary food material to a wide range of animals such as fish, amphibians, reptiles, birds and mammals (Edwards & Bohlen, 1996). The presence of plant and animal growth-promoting amino acids and other substances showed the therapeutic effects (Edwards & Bohlen, 1996). Presently, there is an increasing demand and interest in naturally produced medicines, accompanied by modern laboratory investigations into pharmacological properties of bioactive ingredients and their ability to treat various diseases (Smita, 2021). Further it encourages looking for other molecules of curative value.

In recent years, zootherapy (the science of treating various ailments by using therapeutic materials obtained from animals) is gaining valuable importance as it is potentially safe drugs to use. Therefore, pharmaceutical industries are much interested and are trying to use these bioactive compounds and other metabolites as potent agents in treatment of many diseases (David & Gordon, 2020). Many studies revealed that primary and secondary metabolites are used in the treatment of chronic as well as infectious diseases (Kesava & Usha, 2016). In recent years, GC–MS has become the key tool for profiling secondary metabolites which is widely used in the separation and identification of complex components, as it has high resolution of GC and high sensitivity of mass spectrometry. Hence, the present study is focused on identification of different bioactive compounds through GC–MS technique from the epigeic earthworm, Eisenia fetida, using different solvent system like distilled water, chloroform, methanol and ethyl acetate.

Methods

  1. a.

    Selection and collection of earthworm: Epigeic earthworm, Eisenia fetida species, was selected based on their mass multiplication rate in vermiculture and was collected from the stock culture maintained in vermitechnology laboratory, Department of Zoology, Karnatak University, Dharwad, as it is voracious feeder and breeder continuously and can withstand a wide range of abiotic factors.

  2. b.

    Preparation of sample powder: Around thirty sexually matured healthy Eisenia fetida earthworms were collected and washed them in distilled water and the surface debris were removed with the help of blotting paper. The cleaned earthworms were sundried for about seven days. The dried biomass was ground to get fine powder with the help of mortar and pistil.

  3. c.

    Sample extract: 2.5 g of powdered sample was used and subjected to extraction with chloroform (40 ml), ethyl acetate (40 ml), methanol (40 ml) and distilled water (40 ml) solvents.

  4. d.

    GC–MS analysis: Gas chromatography–mass spectroscopy analysis was carried out to evaluate various metabolites and bioactive compounds present in the powdered sample, the epigeic earthworm, Eisenia fetida, in four different solvent extracts. The sample was analyzed on GC 2010 with split injection mode and linear velocity flow control mode having 800C of column oven temperature with 65.0 kPa of pressure. The GC program was performed on GC–MS–QP 2010 with 2200C Ion source temperature and 2800C of interface temperature. The solvent cut time was 6.50 min with relative type of detector gain mode, and the detector gain is 1.03 kV + 0.20 kV at 1000 threshold. The start time was 7.00 min, and the end time was 50.00 min with scan ACQ mode. The event time is for 0.50 s with 1000 scan speed.

  5. e.

    Identification of components: Various bioactive compounds are identified based on the retention time (R-time). Interpretation of mass spectrum of GC–MS was done by using the database of the National Institute Standard and Technology (NIST). The data reported in the present study are obtained from various online repositories, websites like MedChem Express, PubChem, ChEBI, ChemSpider, Smolecule, Cymitquimicq and Chemical Book. Based on this, the molecular weight, molecular formula, their structure, nature of compounds and their biological activity were tabulated in the results.

Results

The GC–MS profiling of powdered sample of Eisenia fetida in four different solvent extracts (chloroform, ethyl acetate, methanol and distilled water) gave a wide range of bioactive compounds. The GC–MS chromatogram of chloroform extract showed the peaks indicating the presence of 17 bioactive compounds and is presented in Fig. 1. Table 1 indicates the details of identified bioactive compounds such as name of the compound along with its peak number, R-time, base m/z, compound nature, chemical formula and molecular weight, whereas the structure of compounds and their biological activities are presented in Table 2. Similarly, Figs. 2, 3 and 4 present the GC–MS chromatogram of Eisenia fetida which showed the peaks indicating the presence of 22, 21 and 18 different bioactive compounds in ethyl acetate, methanol and distilled water extract, respectively. Tables 3, 4 and 5 present the details of the bioactive compounds such as compound’s name with its peak number, R-time, base m/z, compound nature, chemical formula and molecular weight, whereas the structure of respective compounds and their biological activities are presented in Tables 6, 7 and 8 with respect to ethyl acetate, methanol and distilled water extracts, respectively.

Fig. 1
figure 1

Chromatogram of GC–MS in chloroform extract of the epigeic earthworm, Eisenia fetida

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Table 1 Details of bioactive compounds analyzed in chloroform extract of the epigeic earthworm, Eisenia fetida, through GC–MS
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Table 2 Structure and bioactivity of the compounds obtained in the chloroform extract of the epigeic earthworm, Eisenia fetida, through GC–MS
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Fig. 2
figure 2

Chromatogram of GC–MS in ethyl acetate extract of the epigeic earthworm, Eisenia fetida

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Fig. 3
figure 3

Chromatogram of GC–MS in methanol extract of the epigeic earthworm, Eisenia fetida

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Fig. 4
figure 4

Chromatogram of GC–MS in distilled water extract of the epigeic earthworm, Eisenia fetida

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Table 3 Details of bioactive compounds in ethyl acetate extract of the epigeic earthworm, Eisenia fetida, through GC–MS
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Table 4 Structure and bioactivity of the compounds in the ethyl acetate extract of the epigeic earthworm, Eisenia fetida, through GC–MS
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Table 5 Details of bioactive compounds in methanol extract of the epigeic earthworm, Eisenia fetida, through GC–MS
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Table 6 Structure and bioactivity of the compounds in the methanol extract of the epigeic earthworm, Eisenia fetida, through GC–MS
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Table 7 Details of bioactive compounds in distilled water extract of the epigeic earthworm, Eisenia fetida, through GC–MS
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Table 8 Structure and bioactivity of the compounds in the distilled water extract of the epigeic earthworm, Eisenia fetida, through GC–MS
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GC–MS analysis of different extracts of the epigeic earthworm, Eisenia fetida, showed various bioactive compounds of pharmaceutical importance. There are in total 78 different bioactive compounds identified from four solvent extracts (chloroform, ethyl acetate, methanol and distilled water), which are chemically alkanes, esters, phenol, alkenes, fatty alcohol, carboxylic acid, quinoline, sulfur allotrope, fluorides, alkyl iodides, salt, alkyl benzene, dicarboxylic acid, aromatic compounds, phytosterol, acyclic monosaturated diterpene alcohol, halides, heterobicyclic compounds, cyclicalkanes, lactum and liquid alkane in nature. Out of 78 compounds identified, the biological activities of 23 compounds are not yet reported. The presence of all these bioactive compounds some or other way showed various pharmaceutical properties. Majority of the compounds obtained in the present study are chemically alkane in nature, exhibiting different biological activities like anti-fungal, anti-bacterial, anti-cancer, anti-pyretic, anti-helminthic, anti-inflammatory, etc. biological activities. Sulfurous acid, pentadecyl 2-propyl ester is chemically ester in nature, and exhibits anti-oxidant, anti-microbial and anti-inflammatory activities (Patil & Deshmukh, 2015). 2-Propenoic acid, pentadecyl ester is also chemically ester in nature helps in hydroxylation of liver enzymes, promotes hair growth, and inhibits the production of uric acid and arachidonic acid in the human body (Shunmugapriya et al., 2017). (E)-Phytol, which is chemically phytosterol in nature, exhibits anti-histomal, anti-nociceptive, anti-oxidant, anti-inflammatory, anti-allergic and anti-microbial activities (de Josué et al., 2014). 3,7,11,15-Tetramethyl-2-hexadecen-1-ol, which is chemically acyclic monosaturated diterpene alcohol in nature, exhibits anti-microbial and anti-inflammatory activities (Rajeswari et al., 2012). The aromatic compound, phenol, 3,5-bis(1,1-dimethylethyl)-, which exhibits anti-oxidant and anti-bacterial activity (Rizvi et al., 2014). The fatty alcohol compound, 2-octyldodecan-1-ol, also exhibits anti-inflammatory, anti-oxidant and anti-microbial activities (Cymitquimicq.com). The dodecane, 1-iodo which is chemically alkyl iodide in nature exhibits anti-diabetic activity (Mahmood et al., 2020). Quinoline, 1,2-dihydro-2,2,4-trimethyl- is chemically quinoline in nature exhibits anti-oxidant property which is also used in styrene–butadiene and nitrile–butadiene rubber and latex (Chemical Book). The sulfur allotrope, octathiocane, exhibits anti-cancer, anti-oxidants and anti-microbial activities (Smolecule). The fatty alcohol, celidoniol, deoxy-, exhibits anti-bacterial (Köse et al., 2016) and anti-inflammatory activity (Zakariaa et al., 2014), which helps in chemical communication in Anopheles stephensi mosquito (Brei et al., 2004) and acts as pheromone of Orgyia leucostigma (Grant et al., 1987). The anti-inflammatory activity is also reported in undecane which is chemically liquid alkane in nature and heptadecyl acetate which is chemically ester in nature (PubChem). 2-Piperidinone which is chemically lactum in nature exhibits anti-microbial activity (Dawood et al., 2019). The acyclic alkane, hexane, 3,3-dimethyl-, has been used in the production of phytochemical compound effective in the removal of heavy metals (Ghorbani et al., 2022). The carboxylic acid, hydrocinnamic acid, exhibits anti-oxidant (Razzaghi-Asl et al., 2013), anti-inflammatory (Nagasaka et al., 2007) and anti-microbial activities (Taofiq et al., 2017). Cyclopentane, 1,1,3-trimethyl- is a alicyclic hydrocarbon and exhibits anti-cancer activity (Alaqeela et al., 2022). The anti-cancer, anti-microbial, anti-diabetic, anti-convulsant, anti-inflammatory, anti-viral and anti-tubercular activities are also reported in benzothiazole, 2-(2-hydroxyethylthio)-, which is a heterobicyclic compound (Ruhi & Nadeem, 2013).

Discussions

Many of these bioactive compounds obtained in the present study through GC–MS are commonly reported in various medicinal plants, which are pharmacologically important. Some of the bioactive compounds like hexadecane, tetradecane, eicosane, nonadecane, phytol and tetracosane are identified in the present study which are also identified in the leaf extract of Waltheria indica Linn. exhibiting similar pharmacological activities (Prabhanna & Jayaraj, 2017). Indra et al., (2018) identified several bioactive compounds in Datura starmonium of which docosane, 1,2-benzenedicarboxylic acid, nonadecane and eicosane are identified in the present study with similar biological activity. Tetratriaconitane, pentadecane and quinoline are also identified in the present work which are present in the fenugreek seed oil with similar biological activity reported by Sweeta et al. (2019). Delicia and Shyam (2008) reported the diverse bioactive compounds in the Bombyx mori gut, of which celidoniol deoxy, 2-bromotetradecane, tetracosane, pentadecane, nonadecane and eicosane were also noticed in the Eisenia fetida extracts of the present study exhibiting similar pharmacological activities. Many such GC–MS analyses of phytochemicals have been focused on plant extracts, and very few studies are available on animal tissue extracts. GC–MS analysis to investigate anthelminthic activity using Corallocarpus epigaeus extracts using four different organic solvents against Pheretima postuma earthworm was reported by Kalpesh and Priya (2020) where different bioactive compounds in treating helminthiasis were identified. GC–MS-based approach to measure the response of earthworm to the exposure of carbofuran in the soil was reported by Mohana et al. (2013).

Thus, various chemical compounds obtained from the epigeic earthworm, Eisenia fetida, in different solvent extracts exhibit a wide range of medicinal/biological properties, which can be life savior and can be used in preparation of new drugs to treat a wide variety of diseases in pharmacological industries.

Conclusion

GC–MS analysis of the powdered extracts of the epigeic earthworm, Eisenia fetida, in four different solvent extracts revealed the presence of various biologically active compounds, which are having important medicinal value. In total, 78 compounds were obtained from four different solvent extracts, out of which 17, 22, 21 and 18 bioactive compounds were analyzed in chloroform, ethyl acetate, methanol and distilled water, respectively.

Maximum compounds were obtained from ethyl acetate (22) followed by methanol (21), distilled water (18) and chloroform (17) extracts. As per the literature, the compounds have shown anti-fungal, anti-bacterial, anti-cancer, anti-pyretic, anti-helminthic, anti-inflammatory, anti-viral, anti-allergic, anti-HIV, anti-malarial, anti-tuberculosis, anti-diabetic, anti-convulsant, anti-chistomal, anti-nociceptive, anti-oxidant properties.

Hence, based on the results of the present study, it can be concluded that the epigeic earthworm, Eisenia fetida, has a potent source of bioactive compounds that can be used in pharmacological activities. Further studies are also essential to isolate, characterize and purify particular active compounds responsible for particular therapeutic activities.

Online repositories/websites used

  • National Institute Standard and Technology (NIST)

  • PubChem

  • CheBI

  • MedChem Express

  • Chemspider

  • Smolecule

  • Chemical Book

  • Cymitquimic.com