Antibacterial and COX-1 Inhibitory Effect of Medicinal Plants from the Pamir Mountains, Afghanistan

Plants used to treat inflammatory ailments, pain, fever and infections in the Pamir Mountains in northeastern Afghanistan, were tested for antibacterial and COX-1 inhibitory activity. Water and ethanol extracts of 20 species were tested for antibacterial activity against two gram positive and two gram negative bacteria. The ethanol extract of Arnebia guttata inhibited Staphylococcus aureus with a MIC of 6 µg/mL. Water and ethanol extracts of Ephedra intermedia and the ethanol extracts of Lagochilus cabulicus and Peganum harmala inhibited Staphylococcus aureus at 0.5 mg/mL,and the P. harmala extract further inhibited the growth of Bacillus subtilis and E. coli, also with MICs of 0.5 mg/mL. Ethanol extracts of Artemisia persica (IC50: 0.5 µg/mL), Dragocephalum paulsenii (IC50: 0.5 µg/mL), Ephedra intermedia (IC50: 3.8 µg/mL), Hyoscyamus pusillus, Nepeta parmiriensis (IC50: 0.7 µg/mL) and Rumex patientia subsp. pamiricus (IC50: 3.5 µg/mL) exhibited COX-1 inhibitory activity. The observed in vitro activities support the use of some of the plant species in the traditional medicine systems of the Pamir Mountains.


Introduction
The Wakhan Corridor, in the Pamir Mountains of North-eastern Afghanistan is populated by the Wakhi and Kyrgyz peoples. This area is one of the most remote and isolated areas in the world and the populations rely almost solely on their local herbal medicine. The Third Danish Pamir Expedition documented plant use by both people. A number of plants used to treat infectious diseases, fever and pain was recorded.
The flora of the Pamir/Hindukush Mountains is in general related to the Tibetan and Central Asian floras [1]. Some of the species, however, have a fairly broad distribution through alpine Eurasia, and are well described. Others are endemic to the Pamir/Hindukush and the investigation of these plants has been neglected. All species considered in this study are adapted to a dry high-altitude steppe-environment or associated with man-made irrigation in Wakhi villages, and are all reasonably common within the Wakhan Corridor.
The present study investigated antibacterial and cyclooxygenase-1 (COX-1) inhibitory activity of plants from the Pamir Mountains.

Testing for Antibacterial Activity
Water and ethanol extracts of 20 species used in the Pamir Mountains for ailments which could be caused by bacterial infections, were investigated for antibacterial activity against two gram positive and two gram negative bacteria. Most of the tested extracts did not inhibit the test bacteria.
Water and ethanol extracts of Ephedra intermedia and the ethanol extracts of Lagochilus cabulicus and Peganum harmala inhibited Staphylococcus aureus at 0.5 mg/mL, and the P. harmala extract further inhibited the growth of Bacillus subtilis and E. coli, also with MICs of 0.5 mg/mL (Table 1). Antibacterial activity of P. harmala has been demonstrated previously [2], the activity is due to harmane-type alkaloids [3]. The best antibacterial activity was obtained with the ethanol extract of Arnebia guttata, with an exceptional low MIC of 6 µg/mL against S. aureus, very close to the value of 2 µg/mL obtained with streptomycin ( Table 1). The water extract of A. guttata did not show activity. When used in the Pamir Mountains, the root material is finely chopped and then fried in oil, and the oil is then applied to cotton wool and inserted in the outer ear against earache. This preparation makes sense as it seems the active compounds are not extracted with water. Arnebia species have been used from Turkey to China to treat various bacterial infections [4,5]. The antibacterial activity of Arnebia species is due to alkannin and derivatives thereof [5]. A. guttata had a strong red color, indicating the presence of alkannin-derivatives, and previously several of such compounds have been shown to be present in the species [6,7].

Testing for COX-1 Inhibition
A number of the recorded uses of the plants indicated that the plants might inhibit the prostaglandin biosynthesis, and thereby act as anti-inflammatories, pain killers or febrifuges. Ten plant species were tested for COX-1 inhibitory activity. Ethanol extracts of Artemisia persica (IC 50 : 0.5 µg/mL), Dragocephalum paulsenii (IC 50 : 0.5 µg/mL), Ephedra intermedia (IC 50 : 3.8 µg/mL), Hyoscyamus pusillus, Nepeta parmiriensis (IC 50 : 0.7 µg/mL) and Rumex patientia subsp. pamiricus (IC 50 : 3.5 µg/mL) exhibited the best COX-1 inhibitory effect (Table 2).   An in vivo study on Ephedra intermedia has shown that a methanol extract inhibited swelling in the carrageenan-induced paw edema assay [8]. Bioassay-guided isolation identified ephedroxane as the anti-inflammatory compound [9]. Water extracts of Rumex patientia have in previous studies shown anti-inflammatory activity in several paw-oedema models [10], and also exhibited analgesic effect in formaldehyde-induced pain [11]. Previous analysis of the essential oil of Nepeta pamiriensis collected in this study, showed that the oil contains 98% 1,8-cineole [12]. 1,8-Cineole has in several studies been shown to possess anti-inflammatory activity, including activity mediated via inhibition of the prostaglandin synthesis [13].

Plant Material
Plants were collected during the summer season of 2010 in the Wakhan valley, Big and Small Pamir. Plant material was air dried out of sunlight and kept in paper bags. Voucher specimens were identified by Jens Soelberg and deposited at the Herbarium of The Botanical Museum of Copenhagen University (C) and Kabul University Faculty of Science Herbarium (KUFS). See Tables 1 and 2 for voucher numbers.

Extraction for Antibacterial Assay
Dried, powdered material (1 g) of plant material was extracted with 3 mL of water or ethanol for 30 min in an ultrasound bath. The extract was filtered through a filter paper. The extraction procedure was repeated. After filtration the combined ethanol extract was evaporated to dryness under nitrogen, whereas the water extracts were freeze-dried. The extracts were redissolved in DMSO to 100 mg/mL and diluted with Mueller-Hinton broth to a final concentration of 8 mg/mL.

Extraction for COX-Assay
Dried, powdered material (100 mg) of plant material was extracted with 1 mL ethanol for 30 min in an ultrasound bath and filtered through a filter paper. The extraction procedure was repeated. After filtration the extract was evaporated to dryness under nitrogen and redissolved in ethanol to a final concentration of 40 mg/mL.

Cyclooxygenase-1 Assay
The COX-1 assay was performed according to [14] with minor modifications. Fifty µL of COX-1 (Sigma) (75 unit per sample) and 1,250 µL co-factor solution (0.003 g l-adrenaline, 0.003 g reduced gluthatione) and 200 µL Tris-buffer per sample were preincubated for 15 min on ice. Sixty µL of this solution was added to the test solution consisting of 2.5 µL plant extract and 17.5 µL water and preincubated for 10 min at room temperature. 14 C-Arachidonic acid (20 µL) was added to this enzyme-extract mixture and incubated for exactly 10 min in a water bath at 37 °C. The reaction was terminated with 10 µL 2 N HCl. In each test, two types of controls were run (2.5 µL ethanol and 17.5 µL water): backgrounds in which the enzyme was inactivated with HCl before the addition of 14 C-arachidonic acid; and solvent blanks. The COX-1 inhibitor indomethacin was used as a positive control.
Unlabeled prostaglandin carrier solution (4 µL per sample) was added to the reaction mixture. 14 C-prostaglandins synthesized in the assay were separated from unmetabolized arachidonic acid by column chromatography using silica columns. The assay mixture was applied to the column with 1 mL eluent 1 (hexane:1,4-dioxane:acetic acid (350:150:1 v/v/v)) followed by an additional 4 mL eluent 1 to elute the unreacted arachidonic acid. The prostaglandins were eluted into scintillation vials using 3 mL eluent 2 (ethyl acetate:methanol (85:15 v/v)). Four milliliters scintillation fluid (Pico-Flour 15, Perkin Elmer) was added to the vials and the radioactivity was counted after 1 h in the dark in a TriCarb scintillation counter. The percentage inhibition of the extracts was obtained by measuring the amount of radioactivity in the solutions relative to the solvent blank.
The assay was performed in triplicate. Data were fitted into Grafit5 software for estimation of IC 50 -values.
The results indicate that some of the plant species used in traditional medicine in the Pamir Mountains have in vitro activities that support their use.