GC–MS Analysis of an Herbal Medicinal Remedy to Identify Potential Toxic Compounds

October 1, 2014
Hang P. Nguyen, Irene W. Kimaru
Special Issues

Volume 12, Issue 4

In most countries, herbal medicinal products (HMPs) are introduced into the market without proper scientific evaluation or enforced safety and toxicological studies.

In most countries, herbal medicinal products (HMPs) are introduced into the market without proper scientific evaluation or enforced safety and toxicological studies. Consumers can purchase them without a prescription totally oblivious of the potential hazards that HMPs could pose. In this study, the constituents of an unlabeled HMP obtained from a Somalian patient were evaluated. The HMP was attributed to the Commiphora molmol species from Somalia. The HMP is used as a topical antimicrobial rub for wound treatment. Various analytical techniques were utilized to isolate and identify its constituents. Volatile components were isolated using both steam distillation and Soxhlet extraction. The extracts were analyzed using a gas chromatography–mass spectrometry (GC–MS) system with a 5% phenyl-methylpolysiloxane capillary column and electron ionization. Compounds were identified by comparison of their mass spectra with a built-in National Institute of Standards and Technology (NIST) mass spectra library. Many sesquiterpenes related to C. molmol were identified. Only a few compounds not characteristic of C. molmol were detected.

Herbal medicinal products (HMPs) consist of complex mixtures of one or more plants that contain a range of therapeutically active ingredients arising from plant parts or crude plant extracts. The utilization of HMPs in the treatment of certain human diseases has become common place, particularly in developing countries (1). This is a result of the high costs and side effects of most modern drugs and the fact that HMPs are perceived as effective and safer alternatives. Although HMPs are generally assumed to be safe, they could contain toxic and potentially dangerous constituents (2–4). The majority of HMPs are self-prescribed and are used to treat and manage both minor and long-term illnesses. There is reason for concern over their quality because it can affect their effectiveness and safety.

The greater Rochester, New York, area is home to a large refugee population, including Somali and Congolese people from Africa and Burmese people from Myanmar, which is located in Southeast Asia. These groups of people use many different herbal home remedies from their native countries and, in turn, are significant consumers of HMPs. In these countries, HMPs are introduced into the market without proper scientific evaluations and without any enforced safety and toxicological studies. Consumers can purchase HMPs without prescriptions. Some of these HMPs may be contaminated by other plant species, microorganisms, or contaminants such as pesticides and heavy metals, which could be potentially risky to the patient.

The aim of the present study was to isolate and identify the constituents of an HMP to evaluate the chemical ingredients and identify any potentially toxic compounds. The HMP was obtained from a Somalian patient by a health-care provider from a clinic in Rochester, New York. The most significant issue that the health-care provider faced was that the HMP used by the patient was unlabeled and the supplier was unknown. To the health-care provider, this issue raised many safety concerns for the patient. The patient identified the sample as "malmal" for treatment of skin infection, wounds, and colic pain. The HMP was a hard pale-yellow resin attributed to the oleo-gum resin of Commiphora myrrha (Nees) Engl. var. molmol species, a native to northeast Africa (chiefly Somalia). The Commiphora molmol (Nees) Engl. species is composed of small trees and native to northeastern Africa and Southwestern Asia (5). The bark of these trees and shrubs has fragrant resin that is known for its essential oils (6). These oils are mostly known for their medicinal value (5,6).

Globally, C. molmol is one of the most effective herbal medicines for the treatment of sore throats, canker sores, and gingivitis (7,8). It confers many health benefits including treatment of acne, boils, arthritis, wounds, toothaches, and numerous other illnesses (7,8). Many researchers have identified the phytochemicals of resins found in Commiphora species (5,6,9–12) and similarly in C. molmol (5–8,11–13). The phytochemicals found in the resin of C. molmol species are made of a mixture of volatile compounds. Researchers have documented these phytochemicals to consist of monoterpenoids, including α-pinene, camphene, β-pinene, myrcene, and limonene, and sesquiterpenoids, which are generally categorized into these groups: germacrane, eudesmane, guaiane, cadinane, elemane, bisabolane, and oplopane (7,8,11,13). Some of these volatile compounds have been found to exhibit antibacterial and antifungal properties against Gram-positive and Gram-negative bacteria as well as anesthetic and antihyperglycemic properties (5,6).

Gas chromatography coupled with mass spectrometry (GC–MS) is a commonly used technique for separating and identifying the components of complex volatile mixtures. GC–MS can be a valuable tool in natural product research assisting in the separation and identification of isolated components. Plant essential oils are volatile and well suited to GC–MS analyses. GC is known for its high-resolution separation of structurally similar sesquiterpenes, which are the main constituents of plant essential oils. The use of electron ionization (EI) in MS produces distinctive mass spectral fragmentation patterns enabling mass spectra for unknowns to be searched against libraries of EI spectra to attain identification. It is important that the constituents of the essential oils of C. molmol species be accurately identified. The chemical constituents of these oils can be separated and precisely identified using GC–MS to determine the quality as well as detect the presence of any harmful chemicals. In the present work, the volatile phytochemicals isolated from an unlabeled HMP obtained from a patient and attributed to C. molmol species (Somalian myrrh) from Africa were examined. The phytochemicals were isolated using both steam distillation and Soxhlet extraction. GC–MS was used to separate and identify these phytochemicals and detect any potentially harmful compounds.

Experimental

A resin-based HMP identified as C. molmol obtained from a Somalian patient was provided by a health-care provider from a clinic in Rochester, New York, along with the patient's description of how it should be prepared and administered. Steam distillation was used to extract highly volatile and loosely bound volatile organic components of the resin. Steam distillation was chosen because it is analogous to the boiling method that the patient stated to have used to isolate the essential oil from the resin. The patient informed the health-care provider that to collect the oil, they have to boil the "malmal" and let it cool down before applying the oil to the skin.

The resin (~10 g) was coarsely powdered and extracted using steam distillation and the oil layer was collected from the distillate for GC–MS analysis. Soxhlet extraction was used to extract volatile as well as semivolatile and highly bound volatile organic compounds that could not be isolated effectively using steam distillation. The coarsely powdered resin (~10 g) was extracted using ethyl acetate for 48 h. The ethyl acetate extract was collected and concentrated using rotary evaporation to get a brown mass. The concentrated extract was dissolved in the minimum amount of ethyl acetate for GC–MS analysis.

GC–MS analysis was carried out using a Thermo Fisher Scientific Trace 1300 ISQ single-quadrupole GC–MS system with a built in National Institute of Standards and Technology (NIST) library. The GC–MS system was equipped with a 30 m × 0.25 mm, 0.25-μm df HP-5MS UI (5% phenyl)-methylpolysiloxane capillary column (Agilent Technologies, Inc.). To separate the extracts, the capillary column temperature was programmed as follows: 40 °C for 2 min; ramped at 10 °C/min to 140 °C at a step size of 20 °C with a 3-min hold at each consecutive step; ramped to 200 °C at a step size of 10 °C; and maintained at the same rate and hold time with a final temperature ramp to 260 °C with a 2-min hold time at 220 °C and 250 °C. The injector temperature was set at 220 °C. The carrier gas used was helium at a flow rate of 1.5 mL/min. The concentrated extracts were injected with a 1-μL volume using a split (47:1) injection approach with a split flow of 70.1 mL/min. The instrument was operated using an electron ionization source with the mass transfer line and ion source temperatures set at 250 °C and 220 °C, respectively. The MS data were collected from m/z 30 to 600 with a solvent cutoff of 2 min. Data acquisition and processing was performed using XCalibur software (Thermo Fisher Scientific). The separated components were identified through mass spectral comparison with a built in NIST 11 MS library for XCalibur (Thermo Fisher Scientific).

Figure 1: Total ion chromatogram for the steam distillation extract of C. molmol resin.

Results and Discussion

In this study, steam distillation of the HMP attributed to C. molmol resin produced colorless viscous oil that gave a characteristic myrrh odor. This oil and the ethyl acetate extract obtained through Soxhlet extraction of the resin were both individually analyzed by GC–MS. The GC–MS total ion chromatograms (TICs) of the colorless oil and ethyl acetate extract are presented in Figures 1 and 2, respectively. GC–MS analyses of the extracts led to the identification of 60 compounds. These compounds are listed in Tables I and II according to their elution order from the capillary column. The extracts contained a complex mixture consisting of a high proportion of sesquiterpenes, whereas the fraction of monoterpenes was small. This is in line with the fractions of phytochemicals documented for C. molmol by El-Ashry and colleagues (7), Hanuš and colleagues (11), Shen and colleagues (8), and Morteza-Semnani and colleagues (13).

Figure 2: Total ion chromatogram for the ethyl acetate Soxhlet extract of C. molmol resin.

Research has shown that sesquiterpenes are responsible for antimicrobial activities, smooth muscle relaxing, and analgesic effects (8). The resin evaluated in this study contained one sesquiterpene, isogermafurene or curzerene, which is known to have analgesic effects (14). Hossain and colleagues (15) have documented that the sesquiterpenes α-cadinol, caryophyllene oxide, spathulenol, β-guaiene, and ledene oxide-(ΙΙ) exhibit important antifungal activities. These sesquiterpenes were also identified in the resin evaluated in the present study. Limonene was the only monoterpene identified in the present investigation. Other constituents identified in this work included two lactones — deoxysericealactone and benzofuranacetic acid — as well as two fatty acid methyl esters — eicosapentanoic acid methyl ester and docosahexaenoic acid methyl ester. Other free fatty acids were also identified, including methyl 6,9-octadecadienoate and eicosatriynoic acid. In a study by Ammar and colleagues (16), such fatty acids were also detected in the phytochemical study of Commiphora myrrha also known as C. molmol and attributed to adulteration or bad storage conditions that resulted in the degradation of high molecular weight compounds.

Table I: Compounds isolated from C. molmol resin using steam distillation

Parthenin, a sesquiterpene lactone that is known for its cytotoxicity (17), was also identified in the present study. Parthenin is an active constituent of Parthenium hysterophorus, a weed that is known to cause numerous health hazards (18). The presence of parthenin in the resin could be attributed to the contamination of the resin by the weed, which is very prolific and now is found in Africa, Australia, Asia, and the Pacific islands. A few other compounds that are not commonly found in the resin of C. molmol were also identified in the present study. These include acetonyldimethylcarbinol, isobutyl formate, sec-butyl nitrite, acetic acid butyl ester, dimethylfulvene, isobutyl methacrylate, ethyl-3-propylacrolein, strophanthidol, decamethylcyclopentasiloxane, phenanthrenone, octahydro-naphthalenone, and 1,2,3,4,5,6,7,8-octahydroanthracene, which probably were found because of the contamination of the resin by other plant species and degradation resulting from poor storage conditions or adulteration. A report by C. R. Ganellin and D. J. Triggle (19) shows that acetonyldimethylcarbinol has been isolated from the sleep grass species Stipa vaseyi. Similarly, octahydroanthracene, octahydro-naphthalenone, and phenanthrenone are analogs of polycyclic aromatic hydrocarbons (PAHs) and could potentially be harmful because of the reported toxicity of PAHs (20).

Table II: Compounds isolated from C. molmol resin using Soxhlet extraction

Conclusion

The aim of this investigation was to qualitatively determine the chemical constituents of an unlabeled herbal medicinal remedy obtained from a Somalian patient and attributed to the C. molmol species. Volatile constituents were isolated from the resin-based herbal medicinal remedy using both steam distillation and Soxhlet extraction and were analyzed using GC–MS. Many compounds were detected with the major constituents being sesquiterpenes. These sesquiterpenes were similar to those that have previously been documented for C. molmol species (7,8,11,13). Several of these sesquiterpenes have been reported to show analgesic, anti-inflammmatory, and antifungal activities (14,15). A small number of compounds that are not related to those typically found in C. molmol were identified. The presence of these compounds is indicative of the potential for contamination that can affect the quality, efficacy, and safety of such an unlabeled and unregulated herbal medicinal remedy. This investigation confirms that if extracts of the herbal medicinal remedy obtained from the patient and credited to C. molmol are to be used for medical purposes, safety and toxicity concerns must be taken into account.

Acknowledgments

The authors would like to thank Dr. Asim Abu-Baker, formerly a Professor at the Wegmans School of Pharmacy, St. John Fisher College and Dr. Jennifer Carroll from the University of Rochester Medical Center for providing the herbal medicinal remedy.

Irene W. Kimaru is an Associate Professor at St. John Fisher College in Rochester, New York. Hang P. Nguyen is a student at St. John Fisher College. Direct correspondence to: ikimaru@sjfc.edu

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