All solvents were of analytical grade and obtained from Fisher Scientific (Leicestershire, UK) and Acros Organics (Geel, Belgium). All chemicals and reagents were purchased from Acros Organics or Sigma–Aldrich (St. Louis, MO, USA). The solvents for HPLC were purchased from Fisher Scientific (Leicestershire, UK). RiOS water was prepared by reverse osmosis and water for HPLC was dispensed by a Milli-Q system, both from Millipore (Bedford, MA, USA). Water was passed through a 0.22 mm membrane filter before usage.

Analytical plates for thin layer chromatography (TLC) were silica gel 60 F₂₅₄ plates (20 cm × 20 cm) for normal phase (Merck, Darmstadt, Germany). The spraying reagent p-anisaldehyde was prepared by mixing 0.5 mL p-anisaldehyde (Sigma–Aldrich) with 10 mL glacial acetic acid, 85 mL methanol, and 5 mL sulphuric acid.

Flash column chromatography was performed on a Reveleris iES system from Grace (Columbia, MD, USA) using the Reveleris^® Navigator^TM software. The system is equipped with a binary pump with four solvent selection, an ultraviolet (UV) and evaporating light scattering detector (ELSD) and a fraction collector. The column used was a pre-packed Flash Grace Reveleris silica cartridge (80 g) with a particle size of 40 μm. The ELSD carrier solvent was isopropyl alcohol.

HPLC analysis was carried out on an Agilent^® 1200 series system with degasser, quaternary pump, automatic injection, thermostatic column compartment and a diode array detector (DAD) (Agilent^® Technologies, Santa Clara, CA, USA). A silica based Gracesmart C₁₈ column (250 mm × 4.6 mm, 5 μm) (Grace Vydac, USA) and Phenomenex luna C₁₈ (250 mm × 4.6 mm, 5 μm) (Phenomenex, Torrence, CA, USA) was used together with a suitable precolumn to endure the lifetime of columns.

The isolation of compounds was carried out on a semi-preparative HPLC-DAD-MS system (Waters^®) using a Luna 5 μ (C₁₈) 100A 250 mm × 10.0 mm column (Phenomenex^TM) and Masslynx^® 4.1 software. The system was equipped with HPLC pump 515 (Waters^TM 2767), make-up pump (Waters^TM 511), system fluid organizer (SFO), DAD (Waters^TM 2998), triple quadrupole mass spectrometer (TQD-MS) and automatic fraction collector was used to isolate compounds.

NMR spectra were recorded on a Bruker DRX-400 instrument (Rheinstetten, Germany), operating at 400 MHz for ¹H and at 100 MHz for ¹³C, employing a 3-mm broadband inverse (BBI) probe or a 5-mm dual ¹H/¹³C probe using standard Bruker pulse sequences. DEPT-135, DEPT-90, and two-dimensional NMR (COSY, HSQC, and HMBC) experiments were recorded. In order to assist structure elucidation a ¹³C NMR library was used (NMR Predict version 4.8.57, Modgraph). Deuterated solvents including CDCl₃ (99.8% D), CD₃OD (99.8% D), D₂O (99.9% D) were purchased from Sigma–Aldrich.

High resolution mass spectra was obtained with an Agilent^TM 6530 quadrupole-time-of-flight mass spectrometer (QTOF-MS) equipped with an Agilent^TM Jetstream source. The mass spectrometer was operated in positive and negative ion mode at 20,000 resolution. The instrument was calibrated and tuned with a tune mix (G1969-85000) and during acquisition the accuracy was monitored by using ES-TOF reference mass solution kit (G1969-85001) from Agilent^TM. Mass Hunter^® (Agilent^TM Technologies) software was used for acquisition and processing.

The specific optical rotation was determined on a Jasco P-2000 polarimeter. The samples were dissolved in methanol and optical rotation was recorded at 589 nm with a path length of 50 mm.

UV-VIS (ultraviolet-visible light) absorbance was measured on a Genesys-10UV (Thermoscientific) spectrophotometer. Fluorescence (excitation 335 nm, emission 385 nm, and excitation 370nm, emission 440 nm) was measured on a Tecan^TM Infinite M200 spectrofluorometer.

Kickxia ramosissima whole plant was collected in October 2012 from Takht-e-Nusrati, district Karak (KPK), Pakistan. Afterward the herbarium sheets of the collected whole plant were submitted for identification at the Islamabad Herbarium in the Taxonomy Department, Quaid-I-Azam University, Islamabad, Pakistan, where the voucher specimen was deposited (voucher No. 48 CJ, accession no. ISL-44586). The whole plant was dried under shade followed by powdering and sieving through a 20 mesh filter. All powdered material was stored below 20°C till further use.

Powdered whole plant material (0.955 kg) was extracted with 80% (v/v) methanol by double cold maceration. The extract was filtered through Whatman No. 1 filter paper using a vacuum pump. The collected filtrate was dried using a rotary evaporator under reduced pressure below 40°C. The resultant semisolid material was lyophilised with a final yield of 83.54 g, and stored below 20°C. The liquid–liquid partitioning was performed on the crude extract according to a standard extraction scheme (Figure 1). After partitioning with different solvents as shown in the scheme, n-hexane (1.0 g), methanol 90% (3 g), chloroform (1.69 g), ethyl acetate (1.22 g), n-butanol (9.63 g), and aqueous fractions (67.0 g) were obtained. The collected fractions were dried under reduced pressure at 40°C, lyophilized and stored below 20°C.

The NP (normal phase) TLC for all obtained fractions was performed using various solvent systems as mobile phase, including CH₂Cl₂/MeOH (70:30) with a few drops of NH₄OH and CH₂Cl₂/MeOH (75:25) for the methanolic fraction; CH₂Cl₂/MeOH (78:22) for the chloroform and ethyl acetate fractions; n-hexane/CHCl₃ (93:7) for the n-hexane fraction; CH₂Cl₂/MeOH (25:75 or 36:64) for the n-butanol fraction, and MeOH/CH₂Cl₂ (85:15) for the aqueous fraction, respectively. Developed TLC plates were examined under UV at 254 and 366 nm and after spraying with p-anisaldehyde

An aliquot of 0.8 g from the methanol 90% fraction was dissolved in 2 mL methanol and mixed with 1.1 g silica; the mixture was dried with nitrogen gas. The dried extract was loaded on a pre-packed Flash Grace Reveleris^® silica cartridge of 80 g. The compounds were eluted using a gradient from methylene chloride over ethyl acetate to methanol. Based on UV and ELSD detection, multiple subfractions were collected. All fractions were further analyzed by TLC and similar fractions were combined. In this way 14 subfractions were obtained. Flash chromatography with similar conditions was repeated when necessary. Finally, based on TLC profiling, subfractions KRM2 (200 mg), KRM3(150 mg), KRM5 (210 mg), KRM7 (200 mg), and KRM14 (110 mg) were selected for further HPLC profiling.

Similarly an aliquot of 0.8 g from the ethyl acetate fraction was subjected to flash chromatography as above. The gradient used was from methylene chloride over ethyl acetate to methanol. Based on UV and ELSD detection different subfractions (8 in total) were collected. Finally a total of 4 subfractions was obtained and subfractions KRET2 (210 mg), KRET3 (160 mg), KRET4 (100 mg), and KRET5 (270 mg) were selected for further HPLC profiling.

Likewise an aliquot of 0.8 g from the chloroform fraction was loaded on a flash column as discussed above. The compounds were eluted using a gradient from methylene chloride over ethyl acetate to methanol. Based on UV and ELSD detection different subfractions were collected. All fractions were analyzed by TLC and similar fractions were combined as described above; in this way eight subfractions were obtained. Based on TLC profiling subfractions KRCL1 (135 mg), KRCL2 (125 mg), KRCL4 (160 mg), KRCL7 (150 mg), and KRCL8 (120 mg) were selected further HPLC analysis.

Similarly an aliquot of 0.8 g from the n-butanol fraction was subjected to flash chromatography with a gradient from methylene chloride over ethyl acetate to methanol as previously explained. Finally, 10 subfractions were obtained. Based on TLC analysis, subfractions KRB2 (110 mg), KRB4 (127 mg), KRB5 (131 mg), KRB6 (120 mg), and KRB7 (126 mg) was selected for HPLC analysis.

The chloroform fraction and all obtained subfractions KRCL1, KRCL2, KRCL4, KRCL7, and KRCL8 were analyzed by HPLC using an optimized acetonitrile/H₂O + 0.1% formic acid gradient, ranging from 15% acetonitrile to 80% in 50 min at a flow rate of 1 mL/min. Samples were prepared in a concentration range from 1 to 10 mg/mL in methanol. The isolation of pure compounds was performed by semi-preparative HPLC-DAD-MS using the same gradient at 3 mL/min, yielding compounds 1 (7.5 mg), 2 (5.2 mg), and 3 (5.2 mg).

The ethyl acetate fraction and subfractions KRET3 (250 mg), KRET4 (180 mg), and KRET5 (150 mg) were analyzed by HPLC using an optimized acetonitrile/H₂O + 0.1% formic acid gradient ranging from 15% acetonitrile to 100% in 60 min at a flow rate of 1 mL/min. The isolation of pure compounds was performed by semi-preparative HPLC-DAD-MS using the same gradient at 3 mL/min. Compounds 3 (5.2 mg) and 4 (5.7 mg) were finally obtained.

Similarly the methanol fraction and subfractions KRM2, KRM3, KRM5, KRM7, and KRM14 were analyzed by HPLC using an optimized acetonitrile/H₂O + 0.1% formic acid gradient ranging from 35% acetonitrile to 70% in 50 min at a flow rate of 1 mL/min. The compounds were isolated by semi-preparative HPLC-DAD-MS system using the same gradient at a flow rate of 3 mL/min. Finally compounds 2 (3.3mg), 5 (4.5 mg), and 6 (3.2 mg) were isolated.

The n-butanol fraction and subfraction KRB2, KRB4, KRB6, and KRB7 were analyzed by HPLC using an optimized acetonitrile/H₂O + 0.1% formic acid gradient ranging from 39% acetonitrile to 65% in 45 min at a flow rate of 1 mL/min. Compound isolation was performed by semi-preparative HPLC-DAD-MS system using the same solvent gradient as HPLC at a flow rate of 3 mL/min. Compounds 3 (3.2 mg), 5 (3.2 mg), and 7 (3.8 mg) were finally isolated (Figure 2)

Kickxiasine (1). ¹H and ¹³C NMR, Supplementary Table S1. HR-ESI-MS (positive ion mode), m/z 301 [M+Na]⁺ (calculated 301.1258) consistent with a molecular formula C₁₂H₂₂O₇Na, or C₁₂H₂₂O₇ for 1. UV (acetonitrile/H₂O) λ_max 202, 209, 216, and 225 nm. [α]²⁰_D – 19.072 (c = 0.0011, MeOH).

Mussaenosidic acid (2).¹H and ¹³C NMR, Supplementary Table S2. ESI-MS (positive ion mode), m/z 399 [M+Na]⁺ consistent with a molecular formula C₁₆H₂₄O₁₀. UV (acetonitrile/H₂O) λ_max 237 nm.

Mussaenoside (3). ¹H and ¹³C NMR, Supplementary Table S3. ESI-MS (positive ion mode), m/z 413 [M+Na]⁺ consistent with a molecular formula C₁₇H₂₆O₁₀. UV (acetonitrile/H₂O) λ_max 237 nm.

Linarioside (4). ¹H and ¹³C NMR, Supplementary Table S4. ESI-MS (positive ion mode), m/z 422 [M+Na]⁺ consistent with a molecular formula C₁₅H₂₃O₁₀ Cl. UV (acetonitrile/H₂O) λ_max 210 nm.

Pectolinarigenin (5).¹H and ¹³C NMR, Supplementary Table S5. ESI-MS (positive ion mode), m/z 315 [M+H]⁺ consistent with a molecular formula C₁₇H₁₄O₆. UV (acetonitrile/H₂O) λ_max 275, 335 nm.

Pectolinarin (6). ¹H and ¹³C NMR, Supplementary Table S6. ESI-MS (positive ion mode), m/z 623 [M+Na]⁺ consistent with a molecular formula C₂₉H₃₄O₁₅. UV (acetonitrile/H₂O) λ_max 275, 330 nm.

4-Hydroxy benzoic acid methyl ester (7). ¹H and ¹³C NMR, Supplementary Table S7. ESI-MS (positive ion mode), m/z 153 [M+H]⁺ consistent with a molecular formula C₈H₈O₃. UV (acetonitrile/H₂O) λ_max 226, 287, 338 nm.

Structures of the isolated compounds from Kickxia ramosissima were elucidated using ¹H- and ¹³C-NMR (including DEPT-135 and DEPT-90) and 2D-NMR (COSY, HSQC, and HMBC) spectroscopy (all assignments and spectra are attached as Supplementary Material; Tables S1–S7 and Figures S1–S18, respectively). The molecular ion was derived from the mass spectra obtained with the semi-preparative HPLC-DAD-MS system, and the UV absorption maxima from the diode array detection.

The ¹H-spectrum of compound 1 showed signals due to one methyl, one methylene, six methines, and three methoxyls. The methylene signals at 1.51 ppm (dd, J = 14.0, 8.4) and 2.13 ppm (m) were assigned to H-4. A methine at 4.88 ppm (dd, J = 5.3, 8.4 Hz) was attributed to H-3. A methine signal at 2.05 ppm (d, J = 7.3 Hz, H-9) was coupled with another methine at 4.94 ppm (d, J = 7.3 Hz, H-1), an upfield signal that was indicative of substitution with oxygen. Finally two methine protons showed coupled doublets at 3.77 and 3.93 ppm (J = 4.3 Hz), assigned to H-7 and H-6, respectively. The methyl group at 1.35 ppm (s, H-10) was attached to an oxygen-bearing quaternary carbon as determined by HMBC correlations. The ¹³C-NMR spectrum showed 12 carbon signals. The signals at 98.7 and 98.8 ppm were assigned to carbons C-1 and C-3, respectively. In the cyclopentane ring the signals at 75.9 and 77.3 ppm were attributed to C-6 and C-7, respectively. In the same ring the signals at 83.2, 77.4, and 60.3 ppm were assigned to C-8, C-5, and C-9, respectively. The methyl group (C-10) showed a resonance signal at 17.1 ppm. Three methoxyl groups in positions 1, 3, and 8 showed signals at 50.3, 55.6, and 56.3 ppm, respectively. The protons of these methoxyl groups were correlated in the HMBC spectrum with C-1, C-3 and C-8. The methyl group (C-10) at 1.35 ppm was attached to C-8, which was also established by HMBC correlations of the methyl protons with C-8 and C-9. HMBC (Supplementary Figure S6) correlations from C-1 to the methoxyl group attached to C-8 and to H-9, and from C-6 to H-10 confirmed that the cyclopentane ring was substituted by a methyl group (C-10) and a methoxyl group. Similarly, in the second ring, the signals at 99.7, 99.8, and 40.9 ppm were assigned to C-1, C-3, and C-4, respectively. Also the HMBC correlations from H-1 to the methoxyl group attached to C-3 and from C-8 to the methoxyl group attached to C-1 confirmed the position of these methoxyl groups at C-1 and C-3.

The relative configuration was assigned on the basis of chemical shifts and coupling constants reported for related compounds (Morota et al., 1990; Awale et al., 2005; Ahmad et al., 2006), and by analogy with linarioside (compound 4). The ¹³C-NMR chemical shifts and 2D NMR experiments support the structure of compound 1 as a new compound for which the name kickxiasine was adopted. Kickxiasine is reported here for the first time from nature. Although a number of polymethoxylated iridoids have been reported from different medicinal plants, for instance Tabebuia avellanedae (Awale et al., 2005), Rehmannia glutinosa (Morota et al., 1990; Liu et al., 2014), and Gonocaryum calleryanum (Kaneko et al., 1995), however, the possibility that compound 1 is an artifact formed during extraction and isolation cannot be excluded.

Compound 2 showed typical signals of an iridoid glucoside. Further comprehensive analysis showed that compound 2 was mussaenosidic acid, which has been reported from various medicinal plants including Pedicularis kerneri (Venditti et al., 2016), Kickxia elatine (L.) Dum., Kickxia spuria (L.) Dum. (Handjieva et al., 1995), Lagochilus ilicifolius (Guangzhou et al., 2012), and Vitex negundo (Sehgal et al., 1982). However, mussaenosidic acid is reported here for the first time in Kickxia ramosissima.

Compound 3 was identified mussaenoside, which is the methyl ester of mussaenosidic acid (2). Although this compound has been reported before, still the possibility that it is an artifact formed during extraction procedures in which methanol is used cannot be excluded. Mussaenoside is a common iridoid and has been isolated from a number of plants species, for instance Melampyrum sp. (Damtoft et al., 1984), Bellardia trixago (Ersoz et al., 1998), and Mussaenda incana (Dinda et al., 2005). This is the first report of its occurrence in K. ramosissima.

Compound 4 was identified as linarioside, which was the first example of a chlorine containing iridoid glucoside in nature isolated from Linaria japonica (Kitagawa et al., 1973; Otsuka, 1993). A detailed investigation proved that linarioside is a naturally occurring compound and was not formed as an artifact during the extraction process. Afterward a number of other medicinal plants were reported to contain linarioside. These include Cymbalaria muralis (Kapoor and Reisch, 1974), Asystasia bella (Demuth et al., 1989), Linaria aegyptiaca (Ferhat et al., 2010), and Linaria genistifolia (Ilieva et al., 1992). Here linarioside is reported for the first time in K. ramosissima.

Compound 5 was identified as pectolinarigenin which is a common flavonoid reported from various medicinal plants including Linaria reflexa (Cheriet et al., 2014), Cirsium chanroenicum (Lim et al., 2008), and also from K. ramosissima (Singh and Prakash, 1987).

Compound 6 was identified as pectolinarine, which is glycosylated flavonoid isolated before from a number of plants species including Cirsium japonicum (Liu et al., 2007), Cirsium chanroenicum (Lim et al., 2008), Kickxia abhaica (Al-Rehaily et al., 2006), and K. ramosissima (Ahmad et al., 2006).

Finally compound 7 was identified as 4-hydroxy-benzoic acid methyl ester, occurring in many medicinal plant species, for instance Vitex rotundifolia (Yoshioka et al., 2004) and Houttuynia cordata (Jong and Jean, 1993). This is, however, the first report of its presence in K. ramosissima.

The crude extracts were evaluated for antibacterial and antifungal activities. The highest levels of antibacterial and antifungal activities were observed for n the n-hexane fraction, i.e., an IC₅₀ of 8.0 μg/mL against S. aureus and 24.4 μg/mL against M. canis. None of the other fractions were found active in the test range. Compounds 2–7 were evaluated in an integrated screening panel for antimicrobial activity. Compound 1 was not tested due to the limited amount available. None of the isolated iridoids presented antimicrobial activity in the test range. Although few iridoids have been reported to exhibit antimicrobial activities (Ishiguro et al., 1983; Davini et al., 1986), such activity is surely related to certain structural features. A number of investigations have reported antimicrobial activity of iridoid-rich fractions of medicinal plants, for instance Morinda citrifolia (noni) fruits (West et al., 2012). Nevertheless, iridoids can be considered as prodrugs that may be deglycosylated and activated after oral administration. Flavonoids are well known for their antimicrobial activities (Mohanty et al., 2015; Xie et al., 2015). Also during the current investigation, pectolinarigenin (5) was found active against Staphylococcus aureus (IC₅₀ 49.8 μM). This compound also showed weak antiprotozoal activities against Plasmodium falciparum K1 (IC₅₀ 41.8 μM) and Trypanosoma cruzi (IC₅₀ 32.0 μM). Moreover it was not cytotoxic against MRC-5 cells.

Molecular docking studies for isolated compounds were performed for inhibition of α-glucosidase and soybean LOX (15-Lox) (Table 5). Inside the α-glucosidase active site different binding affinities were determined for the isolated compounds. No good correlation was determined for the binding affinities and in vitro results. However, comparatively less binding affinity was determined for compounds 1 and 7 as compared to other isolated compounds. Lower binding affinities of compounds 1 and 7 are synchronized with the in vitro results. Compound 5 being highly potent against α-glucosidase in vitro, was found computationally to give hydrogen bonding interactions with residues Tyr1251, Gln1372, and Gln1561. The binding pose determined for compound 5 was different from the binding pose of the standard acarbose. For compound 5, a binding affinity of -7.01 kcal mol^-1 was determined in case of α-glucosidase. The putative binding mode of compound 5 inside α-glucosidase is given in Figure 5.

In case of soybean LOX, compound 2 was found to be most potent inhibitor. It was found to bind inside the active site of soybean LOX with a binding affinity of -9.21 kcal mol^-1. Compound 2 gave hydrogen bonding interactions with residues His518, Asp766, and Ile857. Similarly, hydrogen bonding interaction with residue Ile857 was common to hydrogen bonding interaction of the co-crystallized ligand 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid. The putative binding mode of compound 2 inside soybean LOX is given in Figure 6. Based on our findings we could demonstrate the moderate antidiabetic, anti-inflammatory, and antimicrobial activity of some constituents of K. ramosissima and therefore at least in part confirm its antidiabetic use in traditional medicine.