Comparative Antibiotic, Antioxidant, and Anti-Inflammatory Activity of Four Monarda Species

Vicki Abrams Motz, Linda Mull Young, David H. Kinder, Jill Bennett-Toomey, Kelly Hall, Rema Suniga, Alyssa K. Griffith, Rand Abdullatef, Luke Fickenworth, Jacob Thompson, and Jarrod Fair

The Monarda genus (Lamiaceae) has extensive ethnobotanical history as food, antibiotics, analgesics, anti-inflammatories, antioxidants, and anti-cancer treatments Functions have not been considered comparatively along phylogenetic lines, however Monardas have been grouped along evolutionary lines both morphologically and via ribosomal RNA analysis This study compared biological activity of M. citriodora Cerv Ex Lag and M. punctata L (closely related to each other and more closely related to Thymus vulgaris) to M. didyma L and M. fistulosa L (closely related to each other but distantly related to the other two and T. vulgaris). M. fistulosa exhibited greatest zones of inhibition against the respiratory pathogens by Kirby-Bauer analysis (p= 0 .009 by ANOVA) M. citriodora was most effective in blocking LPSinduced inflammation in mouse granulosa cells as evidenced by immunocytochemistry and IL6 assay None of the species tested had anticancer effects against HT29 colorectal cancer cells by XTT testing . M. punctata and M. citriodora had significantly greater antioxidant capability than the others in end of season samples (p =0 .033 by ANOVA), however greatest antioxidant activity was seen in M. fistulosa and M. citriodora in flower (P = 2 0 x 10-20 by ANOVA) . While the presence of some chemical constituents and antibiotic activity did appear to be along phylogenetic lines, reproductive state of the plants, month of harvest, and seasonal rainfall all appeared to impact biological activity .

Monardas are found in all fifty US states and throughout Canada (see Figure 1) The genus Monarda was named for Nicolás Bautista Monardes (1493 –1588), a Spanish botanist and physician who wrote “Dos Libros” in 1569, extolling the therapeutic value of many American herbs, including the Monardas (Boxer, 1963)

All Monardas attract butterflies and bees and are considered important species for bee populations (Wheeler, 2017)

Phylogenetic relationships among Monardas. Thirty-one characters of the genus Monarda were analyzed to ascertain phylogenetic relationships (Scora, 1966) and seventeen species, plus many varieties, were found to fall into two sub genera: Monarda (which contains M. didyma and M. fistulosa) and

Cheilyctis with two sub sections: Cheilyctis (containing M. punctata) and Aristate (containing M. citriodora). These findings were validated by Prather, et al. (2002) using ribosomal RNA They found that, within the Labiatae, the Monarda were genetically well separated from Thymus with M. citriodora being most closely related to thyme, and fairly closely related to M. punctata. But M. fistulosa and M. didyma were very closely related to each other and most distant from thyme

Monarda citriodora Cerv ex Lag (USDA symbol MOCI) (voucher specimen # ONU-HVM-005) (lemon beebalm, purple horsemint) is native to southern states (USDA) (Figure 1); it grows in Ohio as an annual Multiple square stems grow to a height of one to two feet from the base . Leaves are opposite and lanceolate and smell like lemon The flower heads present as whorls in pink to lavender with lighter lavender to white leaf-like bracts beneath . It is found in dry sunny areas of plains and hillsides, preferring sandy loam to rocky soils (Wildflower)

Monarda punctata L. (USDA symbol MOPU) (voucher specimen # ONU-HVM-008) (spotted beebalm, spotted horsemint) is native to the east coast and isolated in CA (USDA) (Figure 1) M. punctata produces multiple square brown to purplish, unbranched stems that grow one half to three feet tall .

The medium green, opposite, narrowly lanceolate leaves have short petioles and smell like Greek oregano Flowers present as densely packed whorls in an arrangement known as a verticillaster . The base color, a creamy yellow is spotted with maroon specks and has large lavender bracts beneath each cluster (Wildflower) .

Monarda didyma (USDA symbol MODI) (voucher specimen # ONUHVM-006) (scarlet beebalm, Oswego tea, red bergamot) is a perennial native of the eastern US including Ohio . Its square green unbranched stems are three feet tall, bearing large, oval, dark green leaves The leaves have a minty aroma . Flowers present as two inch dense, terminal clusters of bright red, narrowly tubular flowers It prefers sun to part shade, and rich, moist, acid soils in open woods and meadows (Wildflower, 2018) .

Monarda fistulosa (USDA symbol MOFI) (voucher specimen # ONUHVM-007) (wild bergamot, beebalm) is a perennial native to most of southern Canada, eastern United States, and northeastern Mexico (USDA) (Figure 1) It grows in clumps of square, deep purplish branched stems, two to four feet tall, bearing large-toothed, oblong, grayish-green leaves that smell minty Lavender to pink or even white twolipped, tubular flowers form dense solitary, terminal heads resting on a whorl of pale, leafy bracts . It prefers sand, loam, or clay soils with sun exposure ranging from full sun to part shade, in dry open woods and marshes (Wildflower, 2018)

The Monardas were widely used to by Native Americans to treat a plethora of conditions M. fistulosa, with the greatest distribution, had the greatest number of uses and was used by the most diverse people (see Table 1 Note: Borders indicate species: Lavender = M. citriodora, Yellow = M. punctata, Red = M. didyma; Purple = M. fistulosa.).

Plant part Use

Group

Preparation

Source

Aerial parts Food Hopi Cooked Fewkes, 1896

Aerial parts Febrifuge

Aerial parts Febrifuge

Aerial parts Febrifuge

Leaf Constipation

Mohegan Infusion Tantaquidgeon, 1972

Delaware Infusion Tantaquidgeon, 1972

Navajo Infusion Vestal, 1952

Ojibwa Decoction Regan, 1928

Aerial parts Stomach Cramps Meskwaki Leaf compound Smith, 1928

Aerial parts Head cold, catarrh Meskwaki Snuff Smith, 1928

Aerial parts Head cold

Leaf Tea, food

Leaf Tea, food

Leaf Analgesic, carminative, Head cold, diaphoretic, diuretic, febrifuge, Heart, Abdominal pain

Root Hemostat

Nanticoke Infusion Tantaquidgeon, 1972

Cherokee - Roberts, 2000

Cherokee - Roberts, 2000

Cherokee Infusion Roberts, 2000

Cherokee - Hamel and Chilotskey, 1975

Leaf Tea Fox Infusion Roberts, 2000

Leaf Tea Ojibwa Infusion Roberts, 2000

Aerial parts Cough, sore eyes, aching kidney, emetic Blackfoot Infusion Hellson, 1974

Aerial parts Wound healing Blackfoot Poultice Hellson, 1974

Flowers Boils Blackfoot Poultice Hellson, 1974

Roots Swollen glands Blackfoot Chewed Hellson, 1974 leaves Cathartic Chippewa Poultice Taylor, 1940 leaves Analgesic, headache Chippewa Poultice Gilmore, 1933

Aerial parts Head cold Chippewa Infusion Gilmore, 1933

Aerial parts Respiratory Aid

Aerial parts Abdominal pain

Leaves Headache, Head cold

Leaves Food

Leaves Hysterics, Heart, colic, flatulence, diuretic, diaphoretic

Roots Stop Bleeding

Aerial parts Toothache, fever, coughs, colds

Leaf Febrifuge

Leaf Chills

Leaf Kidney problems

Crow Infusion Hart, 1992

Dakota Decoction Gilmore, 1933

Cherokee Poultice Hamel and Chilotskey, 1975

Cherokee - Hamel and Chilotskey, 1975

Cherokee Infusion Hamel and Chilotskey, 1975

Cherokee - Hamel and Chilotskey, 1975

Montana Flathead - Hart, 1979

Koasati Decoction

Kutenai Bath Taylor, 1940

Kutenai Infusion Hart, 1994

Leaf Cathartic Choctaw Infusion Taylor, 1940

Leaf Respiratory problems

Choctaw Infusion Hart, 1992

Note: Borders indicate species: Yellow = M. punctata, Red = M. didyma, Purple = M. fistulosa

Chemical composition. Many of the Lamiaceae make essential oils which they store in peltate, glandular trichomes on both leaves and flowers Monardas are divided into two major chemotypes—those in which the predominant constituent is thymol and those in which the predominant constituent is carvacrol (Gwinn et al., 2010) . The plants may release these chemicals as a response to trauma Thymol and carvacrol are found in all the Monardas, additionally p-cymene is high in M. citriodora (Dorman and Deans, 2004) and M. punctata (Li et al., 2014) and has been found in M. fistulosa (Keefover-Ring, 2013) and M. didyma (Mazza et al., 1993) . M. punctata is fairly high in thymoquinone (Johnson et al., 1998); and contains geraniol, linalool, and several terpenes, which are also found in M. didyma (Mazza et al., 1993) .

Antimicrobial efficacy. Both the essential oils and hydrosol of M. citriodora exhibited antimicrobial activity (DiVito et. al., 2021) M. didyma, which is highest in thymol, and M. fistulosa, both demonstrate antibiosis against pathogenic bacteria and fungi with little effect on beneficial microbes (Mattarelli et al., 2017) In M. fistulosa, antifungal activity was attributed to thymoquinone (Inouye et al., 2000), and in M. didyma to thymol (Fraternale et al., 2006) Thus, antimicrobial activity is not directly correlated with thymol concentration implying synergism of thymol with other constituents or possible action of carvacrol

Anti-inflammatory activity. M. fistulosa has demonstrated activity against turpentine-induced inflammation in mice (Zhilyakova et al., 2009) .

Anticancer activity. M. citriodora has been reported to have anti-leukemic activity against HL-60 cell lines (Pathania et al., 2013); however, Katoch et al. (2017) found endophytic fungi on M. citriodora which had anti-cancer effects, potentially the active component of the anti-leukemic effect .

Antioxidant activity. The essential oils of M. punctata trigger the formation of oxidizing free radicals within bacterial cells (Li et al., 2014) M. fistulosa and M. citriodora in flower were both found to be ten times higher in water soluble antioxidants than thyme, dropping to half those levels post flowering (Kharchenko et al., 2015) M. didyma exhibited some antioxidant activities (Fraternale et al., 2006); however, extracts of post flowering leaves of M. punctata and M. citriodora had greatest antioxidant activity (Re et al., 1999)

Vasoactive properties. Thymoquinone (in a synthetic form) is credited with smooth muscle relaxation resulting in lowering of vascular pressure and reducing gut spasticity (Ghayur et al., 2012) .

Plants. M. didyma (plants), M. fistulosa (plants), M. punctata (seeds) and M. citriodora (seeds), were planted in full sun with natural water in raised beds with loamy soil Companion Plants,

Athens, Ohio, supplied all seeds and plants used in this study

Aerial parts were harvested before flowering, in flower, and after flowering (M. citriodora did not bloom in 2018 or 2020; M. punctata did not bloom in 2019) The plants were then dried in a food dryer for 24 hours .

Extracts. Aqueous extracts (in 0 1M pH 4 phosphate buffer) and ethanol extracts were allowed to sit at room temperature for 24 hours and then filtered Acid extracts were boiled 30 minutes in 2M HCl and further extracted with ether to obtain the phenolic fraction, or with ethyl acetate to obtain the flavonoid fraction (Harborne, 1998) All extracts were evaporated and reconstituted to 1 gram of plant material per ml of extract .

Antibiosis. Antibiosis was assessed by Kirby-Bauer disk diffusion testing in which 6 mm diameter disks of filter paper containing extract are placed on the surface of Petri dishes containing Muller-Hinton agar with sheep red blood cells inoculated with cultures adjusted to (1 .5X108 cells/mL), incubated overnight at 37°C with 5% CO 2 The diameters of clear areas around the disks where bacteria did not grow were measured . The greater these zones of inhibition, the greater the antibiotic effect Anticancer. HT29 colorectal cancer cells were deposited onto 96 well plates at 500 cells/ml (100 μl/well), incubated for 24 hours at 37°C, then treated with dilutions of the extracts dissolved in DMSO . Following 48 hr . incubation, the cell viability was assessed via colorimetric tetrazolium salt (XTT) cell viability assay (Cell Proliferation Kit II (XTT), Sigma-Aldrich) .

Antioxidant. Antioxidant analysis was conducted on HT29 cells testing the formation of reactive oxygen species using the protocol outlined in Li et al. (2014) via EnzoROS-ID® Total ROS detection kit

Anti-inflammatory. Granulosa cells from adult C57BL/6 wild type mice were grown in DMEM/F12 for 24 hours at 37°C Cells were either untreated or treated with a dose of 10μg or 100μg of Monarda extract . After 2 hours, 1μg/ml LPS (a known inflammatory agent) was added Cells were incubated for 24 hours at 37°C, fixed, and processed for immunocytochemistry (Bennett et al., 2012) Brown stains indicate inflammation (IACUC approved Protocol Number: 042419) To confirm antiinflammatory activity, pooled, undiluted supernatant from three of these granulosa cell cultures was assayed in duplicate for levels of the inflammatory chemical Interleukin 6 (IL6) using the Mouse IL6 Assay (Sigma-Aldrich)

Vasoactivity. Porcine renal arteries (from abattoir) were suspended in jacketed water baths containing room temperature aerated Krebs solution and contracted with 10 -5 g/ml epinephrine (Sigma Aldrich) . After allowing 30 minutes for tissue rings to stabilize, M. fistulosa essential oil (Rosemary Creek) was diluted in Krebs solution and added to the epinephrine-contracted vascular rings at a concentration of 10 -7 g/ml; and, after stabilization, dosage was increased exponentially to a final concentration of 10 -3 g/ml M. fistulosa essential oil .

Chemistry. In GC/MS analysis of ethanol extracts, thymol, carvacrol, p-cymene, and 3-carene were identified; with thymol and carvacrol peaks overlapping and in greatest amounts In phenolic extracts of end of season plants, distinct differences were found which grouped species along evolutionary lines (Figure 2) . At 3 . 65 minutes, a small peak can be seen in spectra from M. citriodora and M. punctata which is considerably taller in M. fistulosa and M. didyma whereas the converse is true of the peak found at 8 95 minutes . (Figure 2) .

Antibiosis. All Monarda species were more effective against pathogenic microbes than thyme Antibiosis was greatest against the respiratory pathogens S. pyogenes and Klebsiella pneumoniae. All Monarda species exhibited antibiosis against MRSA but were ineffective against the vancomycin resistant gut superbugs, VRE and CRE M. fistulosa had the greatest antibiotic activity both in flower and at end of season . This effect may be one of being best adapted to the environment

M. citriodora, which has the smallest native range, is not native to Ohio and has the least efficacy when grown in Ohio M. didyma and M. punctata have similar ranges—both smaller than fistulosa , relegated predominantly to the east coast, but both native to Ohio M. didyma, which is phylogenetically close to M. fistulosa, has consistently lower antibiotic activity against all microorganisms (Figure 3)

Because it is important to find tools to fight drug resistant infections, Monardas in flower and post flowering were compared to several commercial antibiotics against MRSA M. punctata was as effective as vancomycin and significantly more effective than Ciprofloxacin, Erythromycin, and Cefepime (by ANOVA F (24,11) = 152 3; p = 2 .02 X 10 -19) . It should be noted that only pre-flower M. fistulosa was used in this test, which was later found to have the least antibiotic activity of all M. fistulosa samples (Figure 4)

In M. punctata, which had greatest antibiotic efficacy against MRSA, there were several additional peaks in the GC/MS spectra which have not yet been identified (Figure 5) It is expected that one of these is acting as a synergist to the thymol/carvacrol and would account for the increased efficacy of this extract against MRSA .

Thymol is used by hospitals to disinfect areas with potential exposure to MRSA . Disks were prepared with some of the Monarda constituents to identify a synergist which would enhance the effectiveness of thymol against MRSA or the Streptococci. The flavonoids, luteolin, and quercetin had little antibiosis alone and did not enhance thymol antibiosis . However, thymoquinone—found in M. fistulosa at the same concentration as thymol (Johnson et al., 1998) —greatly exceeded the antibiotic activity of thymol against all streptococcal species tested [S. pneumoniae (lungs), S. pyogenes (skin), and S. agalactiae (vaginal)] as well as against the yeast, Candida albicans (vaginal) . Combined samples had greater efficacy than thymol but less than thymoquinone indicating that the two constituents were not synergists . Thymoquinone is an oxidation product of thymol and carvacrol and is found in many plant species that contain thymol (Darakhshan, 2015) Quinones are scavengers of electrons and react with nucleophiles such as sulfhydryl groups in organic compounds Thus, the mechanism for antimicrobial activity could be ascribed to many different properties of thymoquinone . It is also associated with low toxicity profiles, making it a very attractive compound for development as an herbal therapeutic agent (Adeneye, 2014)

Cymene, characteristically high in Monardas, and found in our M. punctata and M. citriodora samples, has been shown to have synergy with carvacrol against Salmonella typhi (Rattanachaikunsopon and Phumkhachorn, 2009) This too needs to be investigated .

Hot acid/ethyl acetate extracts of M. didyma, M. fistulosa, and M. citriodora before flowering, in flower and after flowering as well as end of season samples of M. punctata (did not flower that year) were tested by Kirby-Bauer analysis . Greatest antibiosis was observed against pathogens S. pneumoniae followed by MRSA; less antibiosis was seen against normal commensal, Lactobacillus acidophilus, and the yeast, C. albicans. Early flowering species, M. didyma and M. fistulosa exhibited only minor change in antibiotic action throughout their reproductive cycles, which occurred during an unusually rainy spring M. citriodora on the other hand, which flowered very late, demonstrated a significant rise in antibiotic efficacy at the end of its growing season, which was typically dry (Figure 6) This serves as a warning to herbalists, as the season during which the herb is collected, as well as production of protective chemicals in plants which flower, has a profound effect on its biological activity; as does the hydration of the plant (Motz et al., 2010)

Anti-inflammatory activity. Extracts of M. citriodora, M. fistulosa, and M. didyma in flower, were found to be non-toxic to cultured mouse granulosa cells at 10 μg/ml but did not prevent lipopolysaccharide (LPS)-induced inflammation (data not shown) At 100μg/ ml, M. citriodora had no apparent proinflammatory effect, however M. fistulosa, and M. didyma induced minor inflammation as indicated by a few dark brown staining granules in the cytoplasm

In all species, treatment with Monardas prior to LPS administration produced lighter staining granulosa cells than the positive LPS control This indicates the ability of Monardas to reduce subsequent inflammation (Figure 7) M. citriodora appeared to have greatest antiinflammatory efficacy . Confirmation of the protective effect of Monarda species was completed by assaying for interleukin 6 (IL6) which is an inflammatory protein known to be stimulated by LPS

All Monarda species prevent LPS induced inflammation (F38,7 = 50 47841, p=2 65E-17) as evidenced by the lower concentration of IL6 found in the supernatant of cells cultured with Monarda species . M. citriodora was better able to prevent LPS induced inflammation than the other Monarda species (P = 0 0276) (See Figure 8)

Anticancer activity. At a dose of 4 g dried herb/ml, no toxicity of any of the Monardas was observed against HT-29 colon adenocarcinoma cells . Thus, no anticancer activity has been demonstrated in this cell line at this concentration .

Antioxidant activity. Early flowering M. didyma (collected during an extremely wet spring) had the least antioxidant activity M. fistulosa and M. citriodora exhibited significantly higher antioxidant activity when plants were in flower during a dry period, and reduced antioxidant activity at the end of the summer Post season samples of M. punctata plants which did not bloom had low antioxidant activity (See Figure 9 )

Vasoactive results: Epinephrine induced contraction was dose-dependent rising from a base contractility set at 0mN to 0 .45 mN/min at 10 -7 mg/ml to 0 .66 mN/ min at 10 -6 mg/ml and 1 04 mN/min at 10 -5 mg/ml . No change in contractility was seen when M. fistulosa essential oil was administered at 10 -7 g/ml or 10 -6 g/ml At 10 -5 g/ml, vascular contraction was