AMERICAN JOURNAL OF UNDERGRADUATE RESEARCH VOL. 6, NO. 3

Extraction, Identification, and Quantification of Harmala Alkaloids in Three Species of Passiflora

Abigail Frye and Catherine Haustein Department of Chemistry Central College 812 University Pella, Iowa 50219 USA

Received: December 17, 2007 Accepted: December 27, 2007

ABSTRACT

Harmala alkaloids are a common plant extract with a number of reported uses including as stimulants and monoamine oxidase (MAO) inhibitors. Their reported activity has led some researchers to identify them as the principal active constituent in passion flowers, an abundant plant which has been identified to have a number of pharmaceutical uses of its own. Harmalas are commonly extracted using chloroform; however, in this case, a green extraction process using ethyl acetate and sodium bicarbonate was applied. Analysis of the harmala alkaloids in Passiflora caerulea, Passiflora incarnata and Passiflora “Coral Glow” was performed quantitatively using the HPLC. Comparison of HPLC results from plant extracts to results from standard solutions concluded that harmol and harmine were present in minor amounts in Passiflora incarnata, harmine was present in large amounts in Passiflora caerulea, and no significant amounts of harmala alkaloids were found in Passiflora “Coral Glow”. The extraction process and HPLC analysis also revealed the presence of the flavonoid derivative Vitexin in Passiflora incarnata. Vitexin was present in relatively large amounts, and as a flavonoid derivative, the compound may have powerful antioxidant activity.

I. INTRODUCTION impediments in the synthesis process made the extraction of the compound from natural Plants can be an excellent source of sources critical, and led researchers to pharmaceutical compounds, but their search for and eventually discover a closely molecules are often complex or difficult to related analogue in the leaves of a synthesize. For example, the chemotherapy European species of ornamental shrub drug paclitaxel, commonly known as Taxol®, called Taxus baccata. Today, Taxol® is an originally discovered in the tree bark of the important drug commonly used to treat Pacific Yew tree called Taaxus brevifolia, is ovarian, breast, and non-small cell lung very difficult to synthesize. The synthesis cancer. process is expensive and laborious. In fact, Another type of plant with since its discovery in the early 1960s, only pharmaceutically significant extracts is three complete syntheses have been carried passion flowers. Of the approximately 500 out. The Holton group and the Nicolaou Passiflora species, Passiflora incarnata has group published their syntheses in 1994 and been the most extensively studied for its the Danishfesky group published an pharmacological effects. Multiple studies additional synthesis in 1996 [1]. The have confirmed that it’s most powerful use is

19 AMERICAN JOURNAL OF UNDERGRADUATE RESEARCH VOL. 6, NO. 3 as a sedative or as an antispasmodic, and often in combination with other drugs. A fluid extract from Passiflora incarnata “significantly prolonged sleeping time” when administered to rats [2]. Passiflora extracts induce a “normal” sleep: patients fall asleep naturally and can be awakened as usual. Additionally, there are no negative side effects. The sedative action of Passiflora is increasingly important in our 24/7 society. Today excessive daytime sleepiness and Figure 1. Basic structure of flavonoids. insomnia affect approximately 60 million Americans [3]. Sleep deprivation and untreated sleep disorders cause an array of still debatable [5-6]. This has caused negative outcomes from poor work researchers to question the importance of performance to car crashes. Although an harmala alkaloids in relation to the average human spends a third of their life therapeutic effects of passion flowers [4]. sleeping and a human being can live longer However, the pharmaceutical importance of without food than without sleep, researchers both flavonoids and harmala alkaloids is not still do not completely understand it [3]. in question. As an antispasmodic, Passiflora The term flavonoids is used to was traditionally used in cases of severe describe a wide variety of compounds found spasm (e.g., tetanus), to abort an epileptic in plants that give them their colorful attack, in children to control the spasmodic pigments. The analysis of these compounds coughing associated with whooping cough, has proven to be valuable in the and to alleviate the spasms that accompany identification of several species of Passiflora. menstrual pain [4]. Bergner [4] also There is strong experimental evidence of indicates that Passiflora extracts are useful flavonoids’ ability to modify the body's in the treatment of the manic phase of reaction to allergens, viruses, and bipolar disorder, the symptoms associated carcinogens. They are perhaps best known with neuralgia and shingles, the fretfulness for their powerful antioxidant activity. Two of teething children, and other general pain. flavonoid derivatives that are typical of Additionally, Passiflora has been used to Passiflora incarnata, are vitexin and control abnormal cardiac arrhythmias and isovitexin. Vitexin is “known to be a potent tension-related asthma, and as an anxiolytic, inhibitor of thyroid peroxidase and also to muscle relaxant, antidepressant, and inhibit fast reacting fibres” [7]. adaptogen [5, 6]. In all cases, Passiflora is Harmala alkaloids are a group of β- especially well-suited for the treatment of carboline alkaloids found in significant pediatric and geriatric patients. It is most amounts in many plant families. These consistent and effective in the treatment of alkaloids have been identified as stimulants the weak, fragile, or exhausted patient. and monoamine oxidase (MAO) inhibitors. Despite the abundance and known Other pharmacological activities such as pharmacological effects of passion flowers, antihypertensive, hallucinogenic, and a relatively small number have been antidepressant effects have been also been properly investigated. Since the late 1960s, attributed to the harmala alkaloids [5-6]. only about 40 species have been Harmine, in particular, was originally used to phytochemically researched [5, 6]. A few treat Parkinson’s disease, but also has been species, notably P. incarnata and P. edulis, found to have vasorelaxant, anti-tumor, and have even been researched extensively, yet anti-HIV effects [8]. More recently, the researchers have not been able to identify antileishmanicidal activities of Harmine have the single active constituent responsible for been studied more closely. Leishmaniasis is their medicinal effects. Flavonoids and a macrophage-associated disease spread alkaloids are most commonly cited as the by the bite of infected sand flies. In the past, active constituents [4-7]. However, the there has been speculation regarding the presence of harmala alkaloids in the pharmacological effects of harmala alkaloids, medicinally significant Passiflora incarnata is and the findings that indicated the

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R = H, OH, OCH3

Figure 2. Basic Structure of harmala alkaloids.

compounds possessed cytotoxic properties. 95 percent of all passion flowers originated However, Splettstoeser et al. [9] report that in the tropical rain forests of South America, harmala alkoloids “have been shown to while the other 5 percent came from North protect neurons against the excitotoxic America, Asia and Australia. They can now effects of dopamine and glutamate.” be found growing wild on six continents and As is the case with Taxol®, harmala many islands worldwide. Although most alkaloids are difficult to synthesize. species thrive in tropical climates, some can However, harmala alkaloids are easy to survive much colder conditions, even extract from species of passion flowers. A temperatures below freezing for extended previous study concluded that one or more periods of time, such as during the winter harmala alkaloid is present in a variety of season [14]. These characteristics make Passiflora species [5-6]. They are the plant easy to grow and consequently an commonly isolated from plant materials by attractive plant to study. extraction with petroleum ether and The three species of passion chloroform [10-11] or methanol often flowers that were used in this study are combined with the Soxhlet extraction [12]. Passiflora caerulea, Passiflora incarnata, Other studies have not focused on the direct and Passiflora “Coral Glow.” P. caerulea extraction of the harmala alkaloids from and P. incarnata usually have blue and passion flowers; however, Lewis developed white flowers, but there is a possibility for an effective extraction method using ethyl variation. The only noticeable difference acetate, which is not as toxic as chloroform between the two species is that P. incarnata [13]. has a wrinklier corona. “Coral Glow” A problem with the extraction of passion vines have deep, rich coral red-pink Taxol® from natural sources is that the flowers that some sources characterize as Pacific yew tree, which was the original hot pink. source, is an environmentally protected species, and is also one of the slowest II. EXPERIEMENTAL growing trees in the world. The isolation process results in killing the tree, and six a. Extraction one-hundred-year-old trees would have to be used in order to extract enough taxol to The basis for the extraction of treat only one patient [1]. Although a closely harmala alkaloids was the acidification of the related analogue called baccatin III has harmalas followed by the removal of been discovered, the extraction and impurities with organic solvent, and following chemical elaboration of the neutralization followed by the removal of the compound to taxol is extremely laborious. harmala into ethyl acetate. However, this is not the case with the The plant extract was obtained by extraction of harmala alkaloids and cutting up the stems, leaves, and tendrils of isoflavonoids from passion flowers. Passion the passion flower plants and allowing them flowers are abundant and the extraction to dry in a dessicator. Approximately 15 process is relatively simple. There are grams of dried plant material was ground approximately 500 known species of using a small coffee grinder and mixed with passion flowers. It is estimated that nearly five times their weight of an acetic acid

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Harmine y = 94538x + 120.27 R2 = 0.9998 25000

20000

15000

Area 10000

5000

0 0 0.05 0.1 0.15 0.2 0.25 Concentration (mg/mL)

Figure 3. Calibration curve for Harmine.

solution containing 30 grams of acetic acid dissolved in a saturated sodium bicarbonate per liter of water. The acetic acid and plant solution and then extracted with ethyl material slurry was stirred for five minutes acetate. In the case of the harmala alkaloid before being filtered using a Bucher funnel standards, approximately one-tenth of a and Whatman 4 filter paper. Two washings gram of standard was mixed with 100 mL of the plant material with the acetic acid sodium bicarbonate solution, and then solution were performed. The aqueous extraceted with 25 mL portions ethyl acetate plant extract solution was washed three four times. In accordance with the times with 50 mL of petroleum ether and 50 procedure used for the plant extracts, mL of ethyl acetate using a seperatory excess sodium sulfate was added to this funnel to remove the organic impurities. The solution before pouring it through a cotton bottom layer was collected and a saturated ball filter and eventually connecting it to the sodium bicarbonate solution was added to rotary evaporator followed by the pump to neutralize the acid. The resultant solution eliminate all liquid solution. The flavonoid was extracted three times with 100 mL ethyl derivatives were for a purely qualitative acetate to remove the aqueous impurities. purpose, so a similar procedure was used The top layer was collected and excess on a much smaller scale, and no quantitative sodium sulfate was added to ensure the measurements were recorded. removal of excess water. The “dry” solution After determining the mass of the was poured through a glass funnel and residue, it was dissolved in methanol for cotton ball filter into a roundbottom flask of HPLC analysis. The plant extract samples known mass to filter out any remaining were passed through a Chrom Tech 0.5 μm solids. The solvent was then removed using disposable filter to remove large particles rotary evaporation using an aspirator. After that would wear down the injection system the resulting residue had been pumped to of the HPLC. dryness, it was weighed. Preparation of the known solutions b. HPLC of the five harmala alkaloids and the two flavonoid derivatives was done in a similar An Agilent 1100 HPLC with UV-vis manner. A known mass of the standard was detection capabilities and a 20 mL injection

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Harmol y = 11579x + 247.84 R2 = 0.9893 2500

2000

1500

Area 1000

500

0 0 0.05 0.1 0.15 0.2 Concentration (mg/mL)

Figure 4. Calibration curve for Harmol.

loop was used. The wavelength used during 1:10 and 1:5 dilutions were made for the the analysis was 340 nm. A Phenomenex harmine standard, and 1:10, 8:50 dilutions Luna column measuring 250 x 4.60 mm and were made for the harmol standard. The with 5u C18 stationary phase was used. concentrations of the dilutions were then The column was heated and maintained at plotted against the area of the resultant peak 25º C with a Phenomenex TS – 130 heater. (see Figures 3 – 4). Elution was done with a methanol water The results are shown in Table 1. gradient at a flow rate of 1.0 mL/min. The The P. incarnata extracts had an average level of methanol was increased from 0% to value of 0.031 mg/g harmol and an average 100% over a ten minute period. This of 0.00935 mg/g harmine. The P. caerulea combination has proven to produce the most samples had an average of 0.098 mg/g consistent results. The HPLC results were harmine. used for identification and quantification of Retention times were used to the harmala alkaloids present in the species identify the compounds present in the of passion flower by comparing known Passiflora extracts. The peaks of the plant solutions to the plant extracts. extracts were compared to the standard peaks. The relative retention times for the harmala alkaloids, harmane, harmine, III. RESULTS & DISCUSSION harmol, and harmolol, and the flavonoid derivatives, isovitexin and vitexin, were Standard solutions of harmine, supported by literature values reported [5-7]. harmol, harmane, and harmolol were The chromatograms Passiflora prepared and analyzed; however calibration incarnata both showed a large peak with curves were only made for harmine and retention times at 3.990 and 3.988 minutes harmol, because they were the only harmala and two smaller peaks at 4.641 and 4.686 alkaloids identified located in the plant minutes and 5.332 and 5.383 minutes. The extracts of P. incarnata and P. caerulea. three peaks were identified as the flavonoid The calibration curves were created by derivative vitexin, and the harmala alkaloids performing the extraction process on a harmol and harmine, respectively. known amount of the standard, and then Abourashad, et. al. [5-6] report finding performing a number of dilutions. A 1:100, measurable amounts of harmane, harmol,

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Passiflora Incarnata Passiflora Caerulea

Concentration of Concentration of Harmine Peak Area Harmine in plant Harmine Peak Area Harmine in plant extract (mg/g) extract (mg/g)

209.59 0.0057 3600.37 0.0935

382.79 0.013 4242.67 0.102

Concentration of Harmol Peak Area Harmol in plant extract (mg/g)

246.79 0.0122

334.82 0.0493

Table 1. Peak areas and the corresponding concentration of harmala alkaloid in the plant extract.

harmine, harmaline, and harmalol in P. HPLC analysis of a standard harmine incarnata plant extract. Grice, et.al. [7] solution. The analyses also showed smaller detected harmane and harmine in P. and broader peaks at 4.321 and 4.431 incarnata extracts along with vitexin and minutes and 4.730 and 4.827 minutes. isovitexin. These peaks could not be identified based Determination of the area of known on comparison with the standard harmala concentrations of standard solutions was solutions of harmane, harmine, harmol, and used to calculate the concentrations present harmolol. in the plant extracts. Two extractions from P. The P. caerulea results were more incarnata were completed and the results reproducible, which following the theory varied greatly. There was an average reported by Grice, et al. [7] that harmala of .031 mg/g of harmol in Passiflora alkaloids levels in passion flowers vary incarnata, with a range of .037 mg/g. There based on when the plant was harvested is to was an average of .00935 mg/g of harmine be expected. The Passiflora caerulea stems, present, with a range of 0.00730 mg/g as leaves, and tendrils used for the extractions well. The largest peak was qualitatively were picked within the same week. Using identified as vitexin. The levels of the the area of the peak, the concentration of harmala alkaloids varied widely between the harmine in the plant extract could be two samples of Passiflora incarnata. This calculated. The average harmine level in was not completely unexpected based on Passiflora caerulea was 0.098 mg/g with a the data reported by Grice, et al. [7] that the range of 0.0085 mg/g. This is much higher levels of harmala alkaloids vary based on level of harmala alkaloid as compared to the the stage of development the plants were levels found in the Passiflora incarnata harvested in. The stems, leaves, and samples. This was an unexpected result tendrils used for the extraction were picked based on the current literature since at different times throughout the year. Abourashad et al [5-6] report higher levels of The chromatograms of the P. harmine in P. incarnata as compared to P. caerulea plant extract both showed a large caerulea. peak at at 5.200 and 5.262 minutes No quantifiable amounts of harmala identified as harmine by comparison to alkaloids were detected in Passiflora “Coral

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Glow.” Abourashad et al/ [5-6] report the 2. Speroni, E. & Minghetti, A. (1988). harmala levels detected in 104 samples of Neuropharmacological Activity of Passiflora, and 50 of the samples had no Extracts from Passiflora incarnata. detectable levels of harmala alkaloids. Planta medica. 488-491. Cavin & Rodriguez [15] state that harmala 3. Weaver, Eris. (2005, January). A good alkaloids frequently occur in such small night’s sleep. Library Journal. 130, 65- concentrations that they have not been 67. quantified, which may be a reason that their 4. Bergner, Paul. (1995, Spring/Summer). presence in species of Passiflora is at times Passion flower. Medical Herbalism: A disputed and not always reproducible. Journal for the Clinical Practitioner 7(1/2):13-15. IV. CONCLUSIONS 5. Abourashad, Ehab, Vanderplank, John, & Khan, Ikhlas. (2003). High-speed The results indicate that P. extraction and HPLC fingerprinting of incarnata and P. caerulea are potential medicinal plants I. Application to sources for harmala alkaloids. For harmine Passiflora Flavonoids. 40 (2), 81-91. production P. caerulea would be the plant of 6. Abourashad, Ehab, Vanderplank, John, choice, and for harmol production, P. & Khan, Ikhlas. (2003). High-speed incarnata would be the best option. extraction and HPLC fingerprinting of However, P. incarnata appears to be even a medicinal plants II. Application to better source for the flavonoid vitexin as harman alkaloids of genus Passiflora. opposed to harmala alkaloids. 41(2), 100-106. P. incarnata is considered the most 7. Grice, I., Ferreira, L, & Griffiths, L. pharmaceutically significant species of (2001). Identification and simultaneous Passiflora; however according to this analysis of harmane, harmine, harmol, research P. Caerulea was the better source isovitexin, vitexin in Passiflora incarnata of harmine and P. incarnata did not have a extracts with a novel HPLC method. large amount of harmala alkaloids. This Jounral of Liquid Chromotography & contributes to the data suggesting that Related Technologies. 24 (16), 2513- harmala alkaloids are not the active 2523. constituent of Passiflora. The relatively 8. Sanchaita, L., Swapan, P., Sibabrata, large peak corresponding to the isoflavonoid M., Santu, B., & Mukul, K. B. (2004, vitexin found in P. incarnata may provide an April). Harmine: Evaluation of its area of future study regarding the chemical antileishmanial properties in various composition and the active constituent(s) of vesicular delivery systems. Journal of Passiflora extracts. Drug Targeting. 12(3), 165-175. Based on the results of this 9. Splettstoesser, F., Bonnet, U., Wiemann, research, it appears that the green M., Bingmann, D., & Busselberg, D. extraction technique using ethyl acetate as (2005). Modulation of voltage-gated opposed to chloroform is a valid method for channel currents by harmaline and the extraction of harmala alkaloids and harmane. British Journal of Pharma- possibly flavonoids. However, further study cology. 144, 52-58. and comparison to results using chloroform 10. Dhwan, K, Kumar, S, & Sharma, A. is necessary to confirm this. Comparison to (2002). Comparative anxiolytic activity past extraction results using chloroform is profile of various preparations of unreliable based on the hypothesis that the Passiflora incarnata Linneaus: A amount of harmalas varies based on the comment on medicinal plants’ stage of development of the plant when it standardization J. Alternative and was harvested. Complemantary Medicine. 8, 283-291. 11. Kartal, M, Altun,S, & Kurucu, S. (2003). REFERENCES HPLC Method for the analysis of harmol, harmalol, harmine and 1. Edwards, N. (1996). Taxol. Retrieved harmaline in the seeds of Peganum November 11, 2005, from harmala L. J. Pharm. Biomed. Anal. 31, http://www.bris.ac.uk/Depts/Chemistry/ 263-269. MOTM/taxol.htm

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12. Xie, J., Lili,Z., & Xu, X.. (2002). (2005). See Affinative separation and on-line www.central.edu/chemistry/cathycv.htm identification of antitumor components 14. Vanderplank, John. (2000). Passion from Peganum nigellastrum by coupling flowers. (3rd ed.). Cambridge, MA: The a chromatographic column of target MIT Press. analogue imprinted polymer with mass 15. Cavin, Janice Clymer & Rodriguez, Eloy. spectrometry. Amal. Chem., 74, 2352- (1988). High-performance liquid 2360. chromotographic identification of simple 13. Lewis, Jeremy, Shriver, James, & carboline alkaloids in specimens of Haustein, Catherine. The free base Heliconiini butterflies. Journal of extraction of harmalas from Syrian Rue. Chromotography. 447 (2), 432-435.

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