Determination of antioxidant and antimicrobial activities of leaf extracts of Otostegia integrifolia
© The Author(s) 2018
Received: 10 November 2017
Accepted: 9 May 2018
Published: 18 May 2018
The extracts from the leaves of Otostegia integrifolia have been reported to show phytochemical analysis, total flavonoid content, antioxidant and antibacterial activities.
Our results revealed that the total flavonoid content of methanol and ethyl acetate extracts is 416.5 + 0.288 and 248.9 + 0.872 mgAAE/100 g respectively. The two extracts also showed good antioxidant activity as well as weak to moderate antibacterial activity against some bacteria.
The leaf extracts from O. integrifolia showed good total flavonoid content, DPPH radical scavenging activity and antibacterial activity. In addition to this, the extracts also showed the presence of some important compounds by phytochemical analysis.
Otostegia integrifolia, more commonly known as Abyssinian rose, a plant belonging to the family Lamiaceae, is endemic to Ethiopia, in the dry evergreen woodlands of the Tigray, Gondar, Wollo, Gojjam, North Shewa, Kaffa and Hararghe regions, as well as in the dry and moist agro climatic zones of the district known as Dega . The plant is also endemic to Eritrea and Yemen . Otostegia integrifolia is a shrub which grows up to 3 m tall, often with paired spines at the nodes. Its leaves are sessile or shortly petiolate. The blade is bluish greyish-green, oblanceolate to lanceolate shaped, and reaches 2–9 cm long [3, 4]. The plant grows in the wild but is also cultivated in gardens. It grows on mountain bush lands and wood lands over grazed slopes at altitudes ranging from 1300 to 2800 m. In Ethiopian traditional medicine, the leaves of O. integrifolia are used for the treatment of several diseases including malaria, for treatment of ophthalmia, as an anti-microbial, antihyperglycemic, and for its anti-oxidant properties used in preventing different kinds of sickness and disorders . In this paper we reported phytochemical analysis, total flavonoid content, antioxidant and antibacterial activities of O. integrifolia leaf extracts.
Results and discussion
The results of phytochemical analysis, total flavonoid content, antioxidant activity and antimicrobial activity tests obtained from different extracts of the leaves of O. integrifolia will be discussed as follows.
Qualitative analysis of phytochemicals present in leaf extracts of O. integrifolia
Determination of the total flavonoid content
Total flavonoid content of leaf extracts of O. integrifolia
Absorbance at 510
mgQE/100 g of dry weight
0.148 ± 0.0009
416.5 ± 0.288
Ethyl acetate extract
0.092 ± 0.0029
248.9 ± 0.872
The antioxidant activities of the extracts of the leaves of O. integrifolia were evaluated by using FRAP and DPPH assays.
Ferric reducing antioxidant power (FRAP) assay
FRAP values of leaf extracts of O. integrifolia (mgAAE/100 g)
FRAP value in mg AAE/100 g dry wt
286.146 ± 0.889
Ethyl acetate extract
219.496 ± 0.566
DPPH radical scavenging activity
DPPH radical scavenging values of leaf extracts of O. integrifolia
DPPH scavenging value (mg AAE/100 g of dry weight)
82.91 ± 0.365
Ethyl acetate extract
32.68 ± 1.545
Comparison of MZI among leaf extracts of O. integrifolia
Extracts and standard antibiotics
Concentration in µg/mL
Average values of zone of inhibition
11.4 ± 0.29
14.6 ± 0.25
8.5 ± 0.09
9..3 ± 0.03
6.4 ± 0.21
11.7 ± 0.37
15.0 ± 0.36
7.6 ± 0.29
12.2 ± 0.33
15.3 ± 0.33
11.1 ± 0.37
10.1 ± 0.04
10.3 ± 0.43
13.5 ± 0.40
15.7 ± 0.26
13.9 ± 0.16
12.2 ± 1.03
Ethyl acetate extract
6.6 ± 0.12
12.6 ± 0.24
11.6 ± 0.34
7.1 ± 0.17
6.9 ± 0.16
13.4 ± 0.34
15.5 ± 0.1
7.8 ± 0.29
14.8 ± 0.26
17.1 ± 0.14
8.6 ± 0.21
9.4 ± 0.49
16.8 ± 0.41
Petroleum ether extract
24.5 ± 0.12
27.7 ± 0.08
25.0 ± 0.12
18.8 ± 0.21
31.4 ± 0.15
16.6 ± 0.17
15.5 ± 0.21
Chemicals and reagents
Ferric chloride (FeCl3), Wagner’s reagent (Iodine in potassium iodide), hydrated aluminum chloride (AlCl3.6H2O), sodium nitrite (NaNO2), hydrochloric acid, sulfuric acid (H2SO4), sodium hydroxide (NaOH), nitric acid (HNO3), sodium carbonate (NaCO3), monosodium hydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), trichloroacetic acid, potassium hexacyanoferrate (II) (K2[Fe(CN)6], Ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), quarticien, ammonia solution, chloroform, acetone, iodine powder, potassium iodide, Muller Hinton agar, ethyl acetate, methanol, petroleum ether, distilled water and deionized water were some of the chemicals and reagents that were used for the experimental work during our study.
Fresh leaves of O. integrifolia were collected from Arebaya, which is located in north Gonder zone and 288 km away from Bahir Dar, Amhara regional state, Ethiopia in May 2017. The plant material was identified and authenticated by Dr. Ali Seidu, botanist in biology department, Bahir Dar University.
Extraction of samples
The air-dried and ground (100 g) of the leaves of O. integrifolia were extracted by soaking successively in n-hexane, ethyl acetate (EtOAc) and methanol (MeOH) each for 24 h (two times with each solvent) and removal of the solvent under reduced pressure using a BUCHI flash evaporator to afford extracts of 2.1 g (for n-hexane), 13.0 g (for EtOAc) and 18.5 g (for MeOH).
The phytochemical analysis of methanol, ethyl acetate and petroleum ether extracts of the leaves of O. integrifolia were studied by slight modifications based on standard procedures described on different literatures [7–10].
Measurement of total flavonoid content
Total flavonoid content was measured with aluminum chloride colorimetric assay as described by different researchers with minor modifications [10, 11]. In brief, 1 mL of methanol and ethyl acetate extracts and 1 mL of standard quercetin solutions (20, 40, 60, 80 μg/mL) were positioned into test tubes and 4 mL of distilled water and 0.3 mL of 5% sodium nitrite solution were added into each solutions. After 5 min, 0.3 mL of 10% aluminum chloride was added. At 6th min, 2 mL of 1 M sodium hydroxide was added and orange yellowish color was developed. The absorbance was measured at 510 nm by using UV–visible spectrophotometer. The blank was performed using distilled water. Quercetin was used as standard. The samples were performed in triplicates. The calibration curve was plotted using standard quercetin. The data of the total flavonoid contents was expressed as mg of quercetin equivalents/100 g of dry mass.
Measurement of free radical scavenging activity
DPPH radical scavenging assay
The antioxidant activity of methanol and ethyl acetate extracts was measured on the basis of the scavenging activity of the stable 1,1-diphenyl-2-picrylhyorazyl (DPPH) free radical according to the method described by Thaiponga et al. with slight modifications [10, 12, 13]. In brief, 1 mL of DPPH solution was added to 4 mL of various concentrations of methanol and ethyl acetate extracts and ascorbic acid to be tested. After 30 min, absorbance was measured at 517 nm. Ascorbic acid with a series of concentration was used as a reference material. All tests were performed in triplicate.
Ferric reducing antioxidant power (FRAP) assay
The reducing power of methanol and ethyl acetate extracts was determined according to the method described by Abebe et al.  with slight modification . In brief, 2.5 mL of different concentration of methanol and ethyl acetate extracts were mixed with 2.5 mL of phosphate buffer solution (PH = 6.6, 0.2 M) and 2.5 mL of potassium hexacyanoferrate ([K3Fe(CN)6]) (1%). The mixture was incubated at 50 °C for 20 min in water bath. Then 2.5 mL of Trichloroacetic acid (10%) was added to the mixture to terminate the reaction. 5 mL of the upper layer of the solution was mixed with 5 mL of distilled water and 0.5 mL of FeCl3 solution (0.1%). The reaction mixture was leave for 10 min at room temperature and the absorbance developed bluish green color was measured at 700 nm by using UV-spectrophotometer against a blank solution. Distilled water was used instead of extracts or standard to prepare a blank solution.
Antimicrobial activities were performed in microbiology laboratory, department of Biology, Bahir Dar University by using agar well diffusion method. Muller Hinton agar media was prepared for culturing selected gram negative and gram positive bacteria by using standard methods. Five bacteria [two gram positive (S. aureus and S. pyogens) and three gram negative (E. coli, S. typhi and K. pneumoniae)] were selected and collected from department of Biology, Bahir Dar University. A series of plant extract concentrations (25, 50, 75,100 μg/mL) and standard antibiotics (Gentamycin and Chloramphenicol) were added to the incubated plate by using filter paper. Then it was incubated for 24 h at 37 °C and the experiment was repeated three times, and average values of zone of inhibition was recorded in mm for antimicrobial activity as described before [14, 15].
The results were reported as mean ± standard deviation (SD). The calibration curves were constructed by using Microsoft excel window 10 and origin 8.
In conclusion, we found that the leaf extracts from O. integrifolia showed good total flavonoid content, good DPPH radical scavenging activity and weak to moderate antibacterial activity. Among those extracts, methanol extract is the one that showed good activities compared to that of ethyl acetate and petroleum ether extracts.
ZYD was supervised the whole work as well as organized the manuscript as a whole and YAC did the experiment. All authors contributed to manuscript finalization. All authors read and approved the final manuscript.
We would like to thank Bahir Dar University for financial support to do this research. Yiketel Adege Chekol also thanks ministry of education, Ethiopia for study leave.
The authors declare that they have no competing interests.
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- Sebald O (2006) Otostegia benth. In: Hadberg I, Kelbessa E, Edward S, Demissew S (eds) Flora of Ethiopia and eritrea from gentianaceae to cyclocheilaceae, vol 5. National Herbarium, Addis Ababa University, Addis Ababa, p 538Google Scholar
- Al-Musayeib NM, Abbas FA, Ahmad MS, Mossa JS, El-Feraly FS (2000) Labdane diterpenes from Otostegia fruticosa. Phytochemistry 54:771–776View ArticleGoogle Scholar
- Ayatollahi SAM, Kobarfard F, Asgarpanah J, Ahmed Z (2007) Chemical constituents of O. perisca. J Chem Soc Pak 29:61–63Google Scholar
- Tofighi ZF, Yassa AN, Goodarzy SR (2009) Chemical composition and antioxidant activity of Otostegia persica essential oil from Iran. Int J Essent Oil Ther 3:45–48Google Scholar
- Sadia NU, Afsar SK, Rizwana SN (2014) Physiochemical screening and antimicrobial potential of Otostegia limbata Benth. J Chem Soc Pak 36:685–688Google Scholar
- Anwar F, Bushra S, Muhammad A (2009) Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts. Molecules 14:2167–2180View ArticleGoogle Scholar
- Osibemhe M, Onoagbe IO (2015) Qualitative and quantitative phytochemical evaluations of Strophanthus hispidus stem bark. J Pharm Biol Sci 10:120–124Google Scholar
- Ram S, Sinha VS (2015) Qualitative phytochemical analysis of some plants uses to cure malaria in Kolhan region of Jharkhand, India. J Med Plants Stud 3:60–62Google Scholar
- Kamala PK, Narendra DS (2014) Studies on qualitative and quantitative phytochemical analysis and screening of in vitro biological activities of Leucas indica (L.) VAR. Int J Herbal Med 2:30–36Google Scholar
- Prashant T, Bimlesh K, Mandeep K (2011) Phytochemical screening and extraction. Internationale Pharmaceuticasciencia 1:99–105Google Scholar
- Rohan SP, Anup SH (2014) Total antioxidant capacity (TAC) of fresh leaves of Kalanchoe pinnata. J Pharm Phytochem 2:32–35Google Scholar
- Enujiugha VN, Talabi JY, Malomo SA, Olagunju AI (2012) DPPH radical scavenging capacity of phenolic extracts from African yam bean (Sphenostylis stenocarpa). Food Nutr Sci 3:7–13Google Scholar
- Abebe A, Abebe M, Mekonnen A (2017) Assessment of antioxidant and antibacterial activities of crude extracts of Verbena officinalis linn root or atuch (Amharic). Chem Int 3:172–184Google Scholar
- Lalas S, Gortzi O, Athanasiadis V, Tsaknis J, Chinou I (2012) Determination of antimicrobial activity and resistance to oxidation of Moringa peregrina seed oil. Molecules 17:2330–2334View ArticleGoogle Scholar
- Naz S, Farooq U, Khan A, Khan S (2014) Physiochemical screening and antimicrobial potentisal of Otostegia limbata benth. J Chem Soc Pak 36:683–686Google Scholar