Phytochemical Constituents, Safety and Efficacy Study of Thymus schimperi and Thymus serrulatus

Background: The genus Thymus is one of the most taxonomically complex genera in the Lamiaceae family and it includes 250350 taxa (species and varieties). T. serrulatus Hochst. ex Benth and T. schimperi Ronniger are the two species of thymus which are endemic to Ethiopia; locally known as Tosign. According to WHO 2018 report, NCDs (non-communicable disease) are estimated to account for 39% of all deaths in Ethiopia and Infectious diseases also represent a continuous and increasing threat to human health in developing countries like Ethiopia. In Ethiopia, 80% of the population use plant remedies or medicinal plants over centuries. Objective: The present study aimed to review Phytochemical-constituents, Safety and Efficacy Study of Thymus schimperi and Thymus Serrulatus. Method: A web-based literature search was done by using scientific databases including Pub Med, Science Direct, Google Scholar and Scopus. Searching was made using key words: Thymus, Thymus schimperi, Thymus serrulatus, phytochemistry, pharmacology, ethnomedicine, efficacy and safety. Results: T. serrulatus grows in Tigray, Gondar, Bale, in the highlands of Semien Shoa and Wollo whereas T. schimperi is widely distributed in Oromia, Amhara and Southern Nations Nationalities & Peoples Regions. T. serrulatus and T. schimperi belong to the chemotypes Carvacrol-Thymol. Carvacrol (63%), thymol (36%-38%), Thymol (49%) are the major constituent of the essential oil of T. schimperi collected from Bale, T. schimperi from Gonder, Shewa & Wello, T. serrulatus from the Tigray region respectively. Treatment of mice with 2000 mg/kg dose of the essential oil of T. schimperi caused 50% mortality indicating that it’s the LD50 is about 2000 mg/kg. However, n-butanol fraction of the aqueous and crude methanol leaf extract T. serrulatus with the oral limit dose of 5,000 mg/kg showed no sign of overt behavioral changes and toxicity where the LD50 were above 10,000 mg/kg. n-butanol fraction and the crude aqueous extract of T. serrulatus has exhibited an increase in urine volume with the highest urine output at the highest dose (1,000 mg/kg) and an appreciable diuretic activity of 94% and 92% respectively. Conclusion and Recommendation: The essential oil of T. schimperi did not cause a rise in serum enzyme level and major organ damage (kidney and liver), but the dose range of 2000 mg/kg causes 50% mortality. The n-butanol fraction of crude aqueous leaf extract of T. serrulatus showed an increase in urine volume with the highest urine output at the highest dose (1,000 mg/kg) and appreciable diuretic activity (94%). Additional chemical isolation, development of dosage form, clinical trial and toxicological study is recommended.


INTRODUCTION
The genus Thymus is one of the most taxonomically complex genera in the Lamiaceae family. It includes 250-350 species and varieties of wild growing evergreen species of herbaceous perennials, subshrubs and aromatic [1,2]. The Thymus species in Eritrea and Ethiopia are restricted to Afromontane and afro alpine regions the species are well known medicinal plants because of their biological and pharmacological properties [3]. Thyme is largely distributed in temperate zones where Ethiopia has considerably abundant Lamiaceae family herb growing at different regions possessing a variety of the wild growing species of this family. which was16% due to cardiovascular diseases and hypertension, is the prominent risk factor for chronic kidney disease, coronary heart disease and ischemic, as well as stroke and hemorrhagic [10]. Infectious diseases also represent a continuous and increasing threat to human health and developing countries like Ethiopia are carrying the major part of the burden. The number of infections caused by new, reemerging or drug resistant pathogens is growing from time to time. So, to fill such gaps in which new antimicrobial agents are urgently needed [11].
80% of the population of Ethiopia use plant remedies or medicinal plants over centuries. Moreover, medicinal plants remain the most crucial or sometimes the only source of therapeutics by playing a key role in the increase and progress of modern studies by being a base for the development of novelties in drugs [12][13][14][15][16]. In indigenous communities like Africa, Plants have played a central part in combating many ailments both in human and livestock. In Ethiopia, the plant based health care endures as the main alternative treatment for different ailments, mainly due to the scarcity of modern medicine, absence of nearby health service stations, expensive prices of conventional drugs for small holder farmers and pastoralists, appearance of drug resistant microbes and/or helminthes and emergence & recurrence of certain diseases [17][18][19].
Many of the modern pharmaceuticals are derived from medicinal herbs. Some of these include digitalis, a heart medication, derived from the Foxglove plant; salicylic acid, the source of aspirin, from Willow bark; and taxol for treating ovarian cancer, from the Pacific Yew tree, quinine and artemisinin are also well known antimalarial drugs derived from the bark of Cinchona tree and Artemisia annua, respectively [20,21]. In Ethiopia, the dried leaves of T. serrulatus and T. schimperi are used as traditional medicine for the treatment of headache, cough, stomachache, earache, liver disease and gonorrhea. They are also used as flavor tea, coffee and different kinds of stew [5].
In Ethiopia T. schimperi possesses antibacterial, anthelmintic and antifungal activity [8,11,22]. It is also one of the most widely used medicinal plants for the treatment of renal diseases, hypertension, inflammation, infections, pain, to wash skin, mouth wash. The essential oil from leaves of T. serrulatus is used as for the treatment of hypertension, renal diseases and used as diuretic, anthelmintic, disinfectant, antispasmodic, carminative, deodorant, diaphoretic, expectorant, sedative, tonic, anti-inflammatory, antimicrobial, antibacterial, antifungal, antiviral, germicidal and libido enhancer [7,8,22]. Historically, In Ethiopia, there are documentations of T. schimperi and T. serrulatus used traditionally for the treatment of different human diseases in different parts of the country. However, the preclinical study such as phytochemical constituents, safety, efficacy and pharmacologic property of T. schimperi and T. serrulatus are not well reviewed in detail from different literature. Therefore, the aim of this study is to review the phytochemistry, efficacy and safety of T. serrulatus and T. schimperi.

Phytochemistry of Thymus schimperi and Thymus serrulatus
Thyme oils contain phytochemicals such as carvacrol, thymol, γ-terpinene, and p-cymene. These phytoconstituents are present in various proportions and characterized by a large amount of monoterpenes which accounts for 90% of the oil. Thymol and carvacrol occur more commonly and accompanied by the couple c-terpinene/p-cymene, the four monoterpenes being biogenetically closely correlated [1,26]. The presence of intraspecific chemotype variation being common in the genus Thymus. Each of the six chemotypes; thymol, carvacrol, geraniol, α-terpineol, thuyanol-4 and linalool are named after its major constituent monoterpene [27,28]. T. serrulatus and T. schimperi belong to the thymol-carvacrol chemotypes [26]. Both the chemical composition and the isolation yield of thyme oils depend on a number of factors, such as the environment or region in which they are grown, the practice in cultivation, the development stage of the plant, harvesting time and habitat [1,6,28,29].
T. schimperi from Bale is composed of carvacrol (63%) as the major constituent whereas T. schimperi from Gonder, Shewa and Wello is composed of thymol (36%-38%) as a dominant phytoconstituent. The essential oil of T. serrulatus collected from the Tigray region had thymol (49%) as the major constituent, similar to T. shimperi which is found around Dinsho [5,6]. However, the essential oil of T. schimperi collected from medicinal market of Merkto was analyzed to contain thymol (56.5%), linalool (18.5%), and carvacrol (8.9%) as major constituents [3]. According to Ermias, et al. [6] the essential oil of T. schimperi which is grown in Addis Ababa is rich in carvacrol (66.2%) and γ-terpinene (13.2%). Destaw, et al. [30] also reported the chemical composition of six essential oils of T. serrulatus and T. schimperi from six localities in Ethiopia, namely Ofla, Alamata, Yilmana Densa, Tarmaber, Butajira and Bale. According to the report of Destaw the major components of essential oil from Ofla are Alamata's thymol (65.6%), thymol (49.6 %), Yilmana Densa's carvacrol (80.4%), Tarmaber's thymol (48.8 %), Carvacrol (71.8%) were the prominent components of the essential oil from Butajira and in the case of Bale were thymol (53.6%). He also studied that T. serrulatus from Alamata and Ofla, T. schimperi from Bale and Tarmaber are of thymol chemotypes. The other two T. serrulatus from Yilmana Densa and T. schimperi of Butajira were found to contain carvacrol chemotypes. T. schimperi and T. serrulatus essential oil contains similar components with other species of thyme, The major components of thymus vulgaris from Romania were thymol (47.59%) and γ-terpinene (30.90%), which indicates that the essential oil analyzed belongs to the thymol chemotype. Carvacrol and thymol were the major compounds found in essential oils of T. capitata and T. zygis from Spain, respectively [27,28,31]. The crude extract and powdered plant leaves of T. schimperi is composed of secondary metabolites; tannins, phenols and saponins as major constituents [32]. The crude extracts and solvent fractions of T. serrulatus leaves have been reported by Amelework, et al. [25] to contain different secondary metabolites. Alkaloids, Saponins, Polyphenols, Tannins and Phytosterols were found in aqueous crude leaf extract of T. serrulatus. Saponins, flavonoids tannins and phytosterols were found in dichloromethane fraction of aqueous crude leaf extracts of T. serrulatus. n-butanol fraction of crude aqueous leaf extract of T. Serrulatus is composed of alkaloids, saponins, polyphenols, tannins and phytosterols which was comparable with the secondary metabolites of hexane, ethyl acetate, chloroform, butanol and methanol leaves extracts of T. vulgaris [33]. Aqueous and 80% methanol crude extract of T. schimperi leaves was qualitatively analyzed and the presence of steroids, alkaloids, flavonoids, saponins and tannins has been detected [34]. The other report showed that the wild Abyssinian thyme aerial parts the flower had highest total phenolic content (0.5 µg/ml) and the leaf the lowest and the whole thyme plant had the highest condensed tannin content (0.9 µg/ml). Distribution of macro and micro nutrients in T. schipmeri Ca (2,776 ± 130 µg/g) was observed to be of the highest concentration followed by Mg (1,786 ± 13 µg/g). From the studied microelements, Fe (728 ± 58 µg/g) was found in a significant amount compared to Mn, Co, Cu, Zn, Ni, and Cd [35,36].

Safety of T. schimperi and T. serrulatus
Kassahun, et al. [37] acute toxicity study on mice revealed that the essential oil of T. schimperi causes depression, shivering, loss of balance and abnormal body movements. Mice treated with 2000 mg/kg dose of T. schimperi oil showed 50% mortality indicating that its LD50 is about 2,000 mg/kg. However, Sub-acute toxicity study of T. schimperi on mice did not cause major organ damage (liver and kidney) and rise in serum enzyme level as compared with the control group plus the ointment prepared from essential oil of T. schimperi which did not show any abnormal skin irritation up on follow up for 14 days. Destaw, et al. [38] also investigated that essential oils of Burning sensations and the most irritation in mice oil was observed during acute oral toxicity study on Alamata (thymol chemo type) oil. The carvacrol chemo-types (Yilmana and Butajira) resulted in reduced growth of mice than did the thymol chemo-types (Ofla, Alamata, Tarmaber, and Bale). The LD50 of the essential oils are in 2,000 µL/Kg to 5,000 µL/Kg dose ranges of body weight of the test mice which was comparable to with the study of other types of thymus species of essential oils of T. algeriensis [39]. Acute toxicity study on 5000 mg/kg oral limit dose of aqueous extract of T. schimperi leaves showed no clinical signs of toxicity, mortality and behavioral changes [32] whereas, n-butanol fraction of the aqueous crude extract and crude methanol leaf extract of T. serrulatus with the oral limit dose of 5,000 mg/kg showed no sign of toxicity and overt behavioral changes [22,25]. Nigatu, et al. [7] report on intra gastric uptake of extracts at doses 300, 2,000, 5,000, and 10,000 mg/kg did not produce any sign of morbidity and mortality in female mice during the acute toxicity study time period and showed that the LD50 of the aqueous leaf extracts of T. serrulatus were above 10,000 mg/kg. He also studied the chronic effect of aqueous leaf extract of T. serrulatus on the general body weight of the mice which showed the overall increment in body weight with time and the concentration of Urea/BUN its level decreased considerably at doses of 200 mg/kg (46.66 ± 2.08) and 600 mg/kg (34.6 ± 3.05) compared with the control groups (53.33 ± 3.05). The study of the gross appearance of internal organs (kidney and liver) of treated mice and rat showed no abnormal changes in shape, texture, size or color compared to the control.
In another report the methanol and aqueous crude extracts of T. schimperi showed no morbidity 14 days (acute toxicity) at dose of 2 g/kg body weight which signifies that the LD50 could be greater than 2 g/kg per body weight in mice [34].

Efficacy of T. schimperi and T. serrulatus
T. serrulatus with 80% methanol crude extract have sown an increase urine volume. Doses of 125 mg/kg and 500 mg/kg were the lowest and the highest urinary excretions with a diuretic index of 1.13 and 1.60 respectively. 88% at 500 mg/kg diuretic activity has been recorded from the extract. T. Serrulatus crude extract from n-butanol fraction of the 80% methanol also increased urine volume and the highest diuretic index (P<0.01) was evident at 1000 mg/kg which was even greater than that of the HCT. The 80% methanol crude extract of T. Serrulatus showed the prominent natriuresis at 500 mg/kg (P<0.001), and 125 mg/kg was the lowest amount recorded. The chloroform fraction of the crude extract did not show significant urinary excretion of both Sodium and Potassium ions. However, showed significant kaliuresis only at 1000 mg/ kg (P<0.05). A dose-dependent increase in the urinary excretion of Sodium and Potassium ions were observed from n-butanol fraction of the 80% methanol extract of T. serrulatus. At the dose of 1000 mg/kg (P<0.001 and P<0.01, respectively) the highest natriuresis and kaliuresis were observed [25].
Amelework, et al. [22] study revealed that the crude aqueous extract of T. serrulatus increased the urine volume at the dose of 250 mg/kg, (1,000 mg/kg) which is the lowest and the highest urine output was recorded respectively, with a considerable diuretic activity of 92 percent in comparison with the standard drug, HCT (hydrochlorothiazide). The dichloromethane fraction of the aqueous extract of T. serrulatus failed to increase in urinary output at 2,50,500, and 1,000 mg/kg doses but, the n-butanol fraction increased the urine output at all test doses (2,50,500, and 1,000 mg/kg). Even the lowest test dose of n-butanol fraction exhibited a significant (P<0.05) diuretic activity of 70% and the highest diuretic activity of the fraction was demonstrated at 1,000 mg/kg, which was about 94%.
According to Bekesho, et al. [8] in vitro vasodilatory effect of aqueous leaf extract of T. serrulatus on thoracic aorta of Guinea pigs. Sequential administrations of relatively low to high concentrations of T. serrulatus aqueous leaf extract (0.5-5 mg/mL) to the organ bath fluid significantly (P<0.001, in all cases) attenuated the force of contractions on thoracic aorta of guinea pigs, which were provoked by KCl in concentration dependent manner both in intact (n=5) and denuded (n=5) endothelium. The percent of aorta relaxation of the T. serrulatus aqueous leaf extract showed a significant (P<0.05) difference between intact and denuded endothelium of the guinea pigs thoracic aorta with a statistical mean of (43.10 ± 0.52)% and (35.60 ± 0.52) % respectively.
Aqueous and methanol 80% extract of T. schimperi showed a reduction in blood glucose levels in a dose-and time-dependent manner. T. schimperi aqueous extracts of 250 and 500 mg/kg showed reduction of 22.65% and 33.15% in plasma glucose levels, respectively after 4 h of extract administration and In case of 80% methanol extraction at 250 and 500 mg/kg showed reduction of 30.06% and 38.35% in plasma glucose levels, respectively [34]. The oral administration of aqueous extract of T. schimperi leaves and its essential oil distillate at doses of (250, 500, 750 and 1000 mg/kg) and (1 and 1.5 ml/kg) was respectively evaluated for J Clin Exp Pharmacol, Vol. 9 Iss. 3 No: 263 OPEN ACCESS Freely available online their diuretic and anti-hypertensive activity against salt-sucrose induced hypertensive rats. The aqueous extract of T. schimperi leaves for all mentioned doses showed positive diuretic activity at 5 hr and the two higher doses significantly increased Na + , K + and Clcontent of urine (Figure 1) [32].
A 50 mg/ml concentration Ethanol, Methanol and Chloroform extract of T. schimperi showed antibacterial activity against standard and clinical isolates of human pathogenic bacteria [40] which was similar pharmacologic activity with other species of thymus [28,41]. The highest MIC values of T. schimpri oil was 0.08 µl/ml against Tricophyton species (scalp isolate), Microsporum and R. rubra species. T. schimperi oil showed activities in the range of concentration from 0.31 µl/ml to 0.63 µl/ml which is comparable with other species of thymus [44]. T. serpyllum essential oil showed the strongest activity in both cases (MIC 2.5-5 µg/mL, MBC 5-10 µg/mL; MIC 1-2 µg/mL, MFC 2-4 µg/mL). T. algeriensis inhibited the growth of selected microorganisms in medium range of MIC 20-80 µg/mL, MBC 40-160 µg/mL (for bacteria) and MIC 5-10 µg/mL, MFC 10-20 µg/mL (for fungi). The essential oils of T. serpyllum, T. algeriensis and T. vulgaris showed significant antibacterial activity, especially against S. mutans, a recognized cariogenic species and their oils also efficiently inhibited the growth of Candida species Shewaye, et al. [3] studied the antifungal activity of the oil of T. schimperi against Aspergillus niger. The essential oil of T. schimperi indicated strong effect on the fungal pathogen (Aspergillus niger) after applying 10 µL/disc of the oil, which completely inhibited up to a diameter of 36 mm and its minimum inhibitory concentration (MIC) was estimated to be lower than 5 µL/disc, which was related with other finding [45]. This is due to the presence of thymol and carvacrol as observed from GC and GC/MS analysis. Awol et al. [11] study, the oil of T. schimperi has shown the greatest inhibition zone diameter of 33 mm against S. epidermidis bacteria and inhibited the growth of two Trichophyton and two Aspergillus species at a concentration lower than 15.75 mg/mL. Higher concentration (23.25 mg/mL) of T. schimperi essential oil inhibits the growth of P. aeruginosa which was comparable with the essential oil of T. kotschyanus species [46].
The oils of T. schimperi and T. serrulatus from different Ethiopian localities had promising protective activity against paracetamol induced hepatic damage in rats. Treatment with 200 µL/kg of the essential oils of T. schimperi (Tarmaber) and T. serrulatus (Yilmana, Alamata) was found to reverse increased levels of AST, ALT and ALP in PAR (paracetamol) treated rats [30].

Pharmacological properties of T. schimperi and T. serrulatus
The oils of T. schimperi possess antifungal and antibacterial activity against different species of fungus and bacteria [3,11,40,43] which was similar with pharmacologic activity of oils of T. hirtus, T. kotschyanus boiss, T. vulgaris, T. mastichina, T. serpyllum and T. algeriensis species [28,41,46,47]. T. schimperi oil had antioxidant and preservative effects [4] and consistent with other species of thymus [2,41,44,46,48,49] and seem to be applicable in both medicine and food industry. The oils of T. serrulatus and T. schimperi from different Ethiopian localities had promising protective activity against paracetamol induced hepatic damage in rats [30].
T. schimperi was one of Ethiopian anticancer plants since it has antioxidant properties [50]. Diuretic activity was observed from the aqueous, 80% methanol crude extract and its different solvent fractions of the leaves of T. serrulatus [22,25] and also vasodilatory effect on KCl induced pre contracted guinea pigs' isolated thoracic aorta rings which might result to antihypertensive effect [8].
T. vulgaris oil showed proliferation inhibition of human cancer cell lines in dose dependent manner [51], T. algeriensis possesses gastro protective effect resulted in maintaining the mucosal integrity and a mild mucosal ulceration at a dose of 117 and 180 mg/kg respectively [39].
T. serpyllum had potent effect on inhibition of human tumor cells lines growth [44]. T. longicaulis, T. pulegioides, T. vulgaris, T. serpyllum, T. striatus and T. praecox also showed anti-acetylcholinesterase activities [2]. All this entire species of thymus had comparable pharmacologic property with T. schimperi and T. serrulatus.

Ethno-medicinal uses of T. schimperi and T. serrulatus
Ethnobotanical study and knowledge of medicinal plants would contribute to improved human health on a local and/or a global level [52]. In west highlands of Ethiopia T. schimperi is one of herbal spices grown in home garden and used as ingredients of mixed spice used in cooking and medicinally important [53] which was consistent with the previous report [54]. T. serrulatus grown in Tigray used as ascaricidal, intestinal paraciticidal and both of T. schimperi and T. serrulatus used for treatment of general pain syndrome, blood pressure, influenza and abdominal pain [53], which was similar to ethnobotanical use of T. serphyllum species in in Pakistan [55]. T. serphyllum used as laxative, a good tonic for the kidney and eye diseases, useful in bronchitis and purify the blood in Sudan [56]. T. schimperi was used not only for human disease, but also for cattle and its flower for honey around Odo Bulu, Bale and Demaro region of Ethiopia [52]. T. schimperi is also used for treatment of circulatory disorder in Ada'a District, East Shewa Zone of Oromia Regional State, Ethiopia [56].
In the overall our ethnobotanical review data in Table 1 reveals that, most Ethiopian community utilized T. schimperi and T. serrulatus for the managements of different types of diseases especially in rural communities of the country.  isolation yield and the chemical composition of the thyme oil are dependent on the environment, growth region, cultivation practices, and the development stage of the plant, habitat and harvesting time. The essential oils of T. schimperi possess antibacterial, antifungal, antioxidant and preservative effects. This review also showed the diuretic and vasodilatory effect of T. serrulatus which was safe and effective. The leaves of T. schimperi was widely used for different ailments at different parts of Ethiopia and more frequently cited by ethno-botanical study than T. serrulatus. Even though, the acute toxicity study of the essential oils of T. schimperi did not cause increase in serum enzyme level and major organ damage (liver and kidney), it causes 50 percent mortality at the dose of 2,000 mg/kg. The n-butanol fraction of crude aqueous extract of T. serrulatus increase urine volume and the highest urine output was observed at the highest dose (1,000 mg/kg) with an appreciable diuretic activity 94%. Further chemical isolation, dosage form development, clinical trial and toxicological study is recommended for both species.