The activity-based fractionation of coffee solutions by a series of chromatography techniques led to the isolation of an active compound I which exhibited a strong inhibitory activity against ?-glucosidase. The structure of compound I was established as norharman (9H-pyrido[3.4-b]indole) on the basis of HR-FAB-MS, 1H NMR, 13C NMR and 1H-1H COSY spectra. Compound I potently inhibited ?-glucosidase in a concentration dependent manner but it did not exhibit any significant activity against other glycosidases. A Lineweaver-Burk plot revealed that its inhibition mode of enzyme was uncompetitive with a Ki value of 0.13 mM.
Keywords: ?-glucosidase inhibitor, ?-carboline, norharman, coffee, uncompetitive inhibitor
Coffee is the most commonly consumed beverages in the world and the health benefits of coffee consumption have been extensively studied : coffee has strong antioxidant properties in vivo [16, 18] and also reduces the risk of Parkinson’s  and Alzheimer’s diseases . Recent studies have demonstrated that habitual coffee consumption is related to a significantly lower risk of type 2 diabetes [17, 19], but it remains unclear what mechanisms and what coffee constituents are responsible for the observed association. Animal and in vitro studies have suggested several plausible mechanisms for a beneficial effect of coffee on glucose metabolism: increase in insulin sensitivity , inhibition of glucose 6-phosphatase , an increase of glucagon-like peptide I concentration , and decreases the rate of intestinal absorption of glucose .
The ?-glucosidase is essential for carbohydrate digestion because carbohydrates must be degraded enzymatically in the intestine before they can be absorbed. The inhibition of ?-glucosidase slows down the process of dietary carbohydrates digestion and avoids postprandial hyperglycemia that plays a central role in the development of chronic diabetes associated complication . Thus, ?-glucosidase inhibitors have exhibited high promise as therapeutic agents for the treatment of metabolic disorders, such as type II non insulin dependent diabetes, obesity, and hyperglycemia .
This work was intended to evaluate ?-glucosidase inhibitory effect of coffee previously reported as hypoglycemic and characterize the active principle isolated from coffee.
Materials and Methods
p-Nitrophenyl (PNP)-?-D-glucopyranoside, PNP-?-D-mannopyranoside, PNP-?-D-glucopyranoside and PNP-?-D-galactopyranoside were purchased from Sigma-Aldrich (St. Louis, MO, USA). Yeast ?-glucosidase, almond ?-glucosidase, E. coli ?-galactosidase, jack beans ?-mannosidase, rat intestinal acetone powders, and norharman were also obtained from Sigma-Aldrich. Unless stated otherwise, all further chemicals were purchased from Sigma-Aldrich. All the reagents were of analytical grade.
The UV spectrum was recorded on a Shimadzu model UV-160 spectrophotometer. High- resolution FAB mass spectra were obtained with a JEOL model JMS-AX505 HA spectrometer. 1H-NMR and 13C-NMR spectra were obtained on a Brucker AV 500 spectrometer operating at 500 and 125 MHz, respectively. (CD3)2CO was used as the solvent.
Enzyme inhibition assay
The intestinal ?-glucosidase inhibitory activity was determined as described previously with a slight modification . The rat intestinal acetone powder was suspended in 100 mM sodium phosphate buffer (pH 7.0) and centrifuged at 12,000 rpm for 15 min. The resultant supernatant was used as the source of the small intestinal ?-glucosidases. For the assay of inhibitory activities of maltase and sucrase, the reaction mixture consisted of crude enzyme solution, 20 mM maltose or 200 mM sucrose, 100 mM sodium phosphate buffer (pH 7.0) and a given amount of inhibitor (50% dimethyl sulfoxide solution) in a total volume of 0.5 ml. After the reaction mixture was incubated for 15 min at 37 â„ƒ, reaction was stopped by heating the mixture at 100 â„ƒ for 5 min. The ?-glucosidase activity was estimated by measuring the liberated glucose amount using the glucose oxidase method. Prior to measuring the glucose amount, the interfering agent, phenolic compounds were removed from reaction mixture by passing through a basic alumina column (1 x 3 cm). Acarbose was used as the positive control.
The enzymatic activities of the various glycosidases were determined spectrophotometrically by monitoring the release of p-nitrophenol from the appropriate p-nitrophenol glycoside substrate . The assay solutions and the potential inhibitors were added to a 96-well plate as follows: 20 L of 0.1 M phosphate buffer (pH 7.0), 20 L inhibitor, 10 L enzyme (1 U/mL), 10 L of 25 mM substrate and 40 L of methanol. Following incubation at 37 °C for 15 min, the assay solution was stopped by adding 300 L of 1 N NH4OH solution. The glycosidase activity was determined by measuring the amount of 4-nitrophenol released from p-nitrophenol glycoside substrate was determined with a microplate reader model 550 (Bio-Rad, CA, USA) at 405 nm.
All of the analyses were performed in triplicate. The concentration of the inhibitor required for inhibiting 50 % of ?-glucosidase activity (IC50) was calculated by adjusting the experimental data (% inhibition versus the concentration of the inhibitor) to non-linear regression curves. The mechanism of enzyme inhibition was assessed by analyzing the double- reciprocal Lineweaver-Burk plot.
Isolation of inhibitory compound from coffee
Filtered brewed coffee was prepared in a household coffee maker: 75 g of ground roast coffee of Columbian Supremo (Arabica variety) and 500 ml water to give a brewed coffee. Commercial instant coffee (Tasters’ choice, Nestle) was made by dissolving 75 g instant coffee in 300 ml of hot water. Filtered brewed coffee and instant coffee solutions were separately centrifuged at 12,000 rpm and room temperature for 15 min, and used for isolation of ?-glucosidase inhibitor. The supernatant was adjusted to pH 9 with 1 N NaOH and extracted with ethyl acetate. The ethyl acetate layer was then extracted with 0.1 N HCl solution. This acidic solution was again adjusted to pH 10 with aqueous ammonia and extracted with ethyl acetate. The organic layer containing basic components was subsequently evaporated in vacuo. Forty batches of the above ethyl acetate extracts (total 3 kg each of ground coffee and instant coffee) were concentrated and subjected to silica gel column chromatography with an isocratic solvent system of chloroform-acetone (70:30). Fractions containing the active compound (F3-F6) were combined, evaporated, and subjected to a Sephadex LH-20 column (3 x 35cm) with MeOH as an eluent. Fraction number 10-12, which showed a high inhibition and a similar TLC profile (silica gel 60 F254, Merck, chloroform:acetone = 1:1, rf 0.2) were combined and further purified. The final purification of the active compound was achieved through semi-preparative HPLC separation on a reversed phase C18 column (?Bondapak, Waters, Milford, MA, USA) eluting with 75 % MeOH and detected through absorption at 254 nm. The retention time was 14.5 min. After removing the HPLC solvent in rotary evaporator, the active compound was obtained as a white powder by crystallization from cold acetone.
Results and Discussion
Both instant coffee and ground brewed coffee solutions inhibit ?-glucosidase enzyme activity. Instant coffee showed a slightly higher degree of inhibition than brewed coffee (Data not shown). The activity-based fractionation of coffee solutions by a series of chromatography techniques led to the isolation of an active compound I (2.24 ?g/ g of roasted ground coffee; 3.85 ?g/ g of instant coffee) which exhibited a strong inhibitory activity against ?-glucosidase.
The isolated compound I was shown to be chromatographically pure by TLC and HPLC with various solvent systems and deduced to be a nitrogen-containing compound based on a positive reaction to Dragendorff’s reagent. The UV spectrum of the compound in methanol exhibited absorption maxima at 230, 285 and 348 nm. The molecular formula of compound I was determined to be C11H8N2 (M m/z 168.0736; calcd. 168.0688) by high resolution mass analysis. 1H NMR spectrum of compound I showed 7 aromatic proton signals (?7.2-8.9 ppm) and one free proton signal (?10.63 ppm). 13C NMR spectrum showed 11 carbon signals around 110-145 ppm (Table 1). Taken together, the structure of compound I was deduced as ?-carboline, norharman (9H-pyrido[3.4-b]indole, Fig. 1) with 1H NMR, 13C NMR, and 1H-1H COSY spectra and confirmed by comparison of physical data with those of the authentic specimen.
Compound I potently inhibited ?-glucosidase in a concentration dependent manner, but it did not display any significant inhibitory effects against ?-glucosidase, ?-mannosidase, and ?-galactosidase when tested at a concentration of 10 mM (Table 2). The inhibitory profile demonstrated that the activity of compound I was greater against maltase compared with sucrase (IC50 values: 0.27 mM for maltase and 0.41 mM for sucrase). Although the inhibitory potency was weaker than that of therapeutic drug acarbose (IC50 value: 0.18 mM for maltase and 0.02 mM for sucrase), observed data clearly indicated the potential of compound I as an ?- glucosidase inhibitor. The pre-incubation of compound I with the enzyme increased the inhibition of ?-glucosidase activity, implying that this compound reacted with the enzyme slowly. The ?-glucosidase activity was fully restored when the enzyme was incubated with an amount of compound I which could inhibit enzyme activity up to 90 % followed by eliminating the compound I with a PD 10 desalting column (Pharmacia, Piscataway, NJ, U.S.A). This result demonstrated that compound I was a reversible inhibitor. A double-reciprocal Lineweaver-Burk plotting under various amounts of compound I showed linear lines intercepting on 1/V axis in parallel. The kinetic data suggested that the compound I was an uncompetitive inhibitor, with a Ki value of 0.013 mM (Fig. 2). As a result, compound I, a reversible uncompetitive inhibitor of ?-glucosidase, was isolated from coffee and identified as an active principle. When compound I was given in combination with a carbohydrate-rich diet orally, the postprandial plasma glucose levels were significantly dropped in non-diabetic rats (unpublished data).
Compound I, a tricyclic indole ?-carboline alkaloid norharman is distributed widely in biological systems and exhibits a wide spectrum of pharmacological and neurological effects: antidepressant and antianxiety effects in rats , inhibitory activities of monoamine oxidase and nitric oxide synthase , as well as an increase of insulin secretion two- to threefold from isolated human islets of Langerhans . However, ?- glucosidase inhibitory activity of norharman has not previously reported. Coffee has been noted as the primary exogenous source of norharman. A high variability in ?-carboline content of coffee samples was observed between coffee species (arabica, robusta) and also depended on roast degree and instant coffee production process. An average of 3 cups of coffee per person per day could account for an ingestion of up to 72 ?g of norharman , although this will depend on the coffee strength.
Coffee contains numerous substances. However, little is known regarding the effects of individual constituents on glucose metabolism. The cohort study has supported that the most prominent coffee compound caffeine is irrelevant to risk of type 2 diabetes . Chlorogenic acid, the most abundant polyphenol in coffee, has been shown to reduce glucose concentrations in rats, caused by increasing insulin sensitivity as well as reducing hepatic glucose output through inhibition of glucose 6-phosphatase . Without excluding any other possible mechanism, this report observes ?-glucosidase inhibitory activity as a possible mechanism of hypoglycemic effect of coffee and assigns ?-carboline alkaloid norharman as one of active principles in coffee. Coffee appears to contain active principles other than norharman as evidenced by several active peaks in chromatography systems. It may be possible that various active constituents in coffee act synergistically against ?-glucosidase activity. Characterization of other active principles is under progress.
Fig. 1. Structure of compound I (?-carboline alkaloid norharman).
Fig. 2. A Lineweaver-Burk plot analysis of rat intestine ?-glucosidase inhibition by compound I.
4-Nitrophenol-?-D-glucopyranoside was used as a substrate. The concentration of compound I was 0 mM () or 0.25mM (). The values are expressed as means of triplicate reactions.
Table 1. 1H and 13C NMR data for compound I in (CD3)2CO (? in ppm and J in Hz)
(1H, br, s)
( 1H, d) J=5.5
(1H, d) J=5.5
(1H, d) J=8.0
(1H, ddd) J=8.0, 7.0, 1.0
(1H, ddd) J=7.5, 7.5, 1.0
(1H, dd) J=8.2, 1.0
(1H, br. s)
Table 2. Inhibitory effects of compound I against various glycosidases
IC 50 ( ?M)
180 ± 3.2
Maltase (rat intestine)
270 ± 4.5
Sucrose (rat intestine)
410 ± 11.3
>1.0 x 104
?-mannosidase (jack bean)
>1.0 x 104
?-galactosidase (E. coli)
>1.0 x 104
Values are expressed as the means of triplicate reactions ± standard deviation.
Impacts of Human Population Growth
The Growth of the Human Population
Since the world’s origin, we have seen a steady growth in the worldwide population. From the first records of humans on this earth we have seen the cycle of birth and death. The population increases and decreases are determined by the number of births compared with the number of deaths over the same period. As we will discover, throughout history we have seen many contributing factor to our population fluctuation. Natural disasters along with man-made decisions are two examples of causes for increases or decreases in our population growth. There has also been advancements in technology, medical treatment, and nutrition. We track population growth in two ways. Absolute growth is the difference in population over time. An example of that would be comparing the population in 1961 to the population in 2000. Relative growth is rate or a percentage of the current population.
Starting with the first population tracking which occurred between 2500BC and 2000 BC we see a flat growth rate until around 1000BC. At that time the world’s population rate was around 300 million. From 1000BC to around the year 1750AD we saw a very slow but positive increase in our population. Our population at that time was around 800 million. Until that time our population was keep mainly in check due to the high death rates from plagues, famines and unsanitary living conditions. After the 1750s we saw a faster growth rate and by the year 1950 we had increased our population to 2.5 billion. The population increase from 1950 to 1985 was even faster giving us a population of over 5 billion in 1985. By the year 2000 we were at a population of 6 billion and by the year 2020 we are expected to be a 9 billon.
As technology has advanced, so has the population. From the creation of tools to the development of agriculture and the later rise of industry, has given additional resources to support an increase in our population growth. With the development of basic technology such as wooden tools for farming or weapons for protection and hunting we began the process of finding better and faster ways with bigger and more advances products. Fire was another advancement , not only could it be used for heat, but light, tool making, and allowed the cooking of food thus reducing some potential life threating disease which came for eating raw meet. Once technology had advanced to the point we could produce enough food and other produces to survive we were able to look at other benefits to advancing the human population such academics. As technology continued to advance we began to see specialize classes emerge. The ability to move to other types of workers such as scribes, metal workers among others helped to create an urban society where people could combine their talents to find even more advanced products to improve people’s and lengthen the lives and increase the population. While it is proven that technology has improved and our lives and contributed to our population growth. In some third world countries there has been a slowing or even a reduction in their population growth. These countries must be brought in line with the industrial countries. The transformation from the industrial age to the information age is just another step forward and in this continues cycle of life.
The advancement in medicine is one of the key reasons we started seeing such a population increase around 1000bc. The ancient Egyptians and Romans were one of the first civilizations the understood the importance of medicine and medical procedures. It is where the first people who treated the ill were called doctors. The first medicines used came for existing plant life. Over many generations families would pass down plants that they would us to treat certain wounds or illnesses. While no one truly understood why such herbals would work they were grateful they did. As we continued to advance in the creation of medicines and the development of medical procedures we saw an increase in the life expectancy of our older population. While many of the first medical procedure were trial and error it was a learning experience that helped to identify better procedures. Over the thousands of years many new medicine were created such as vaccines for polio and many other disease that before the vaccine would have truly mend death to the patient. As medical procedures advance so do deadly illnesses such as AID and cancer. We have made great strides in medicine and medical procedures which has helped the population growth by people living longer and more babies and mothers surviving child birth. Even with all the advances in medicine we still have some countries that have seen a slowing or even a reduction in their population growth.
As our world progressed through out the thousands of years many different cultures were formed. Because of the advancements in transportation different cultures began migrating to trade products and to exchange ideals. It was during this change in ideas where we able to blend cultures and learn from each other. The technology of one culture was shared with another thus advancing the world and improving the lives of all. This caused the population grow and structures to be created that help keep order and bring laws thus reducing wars. It also helped to identify what land belonged to whom and ensured that those boundaries were respected. Cultures differences could also have a negative impact on the population. If a culture for example has a belief that a medical procedure is wrong or a certain food is forbidden then that could affect the health of their people thus shorting their lives. If a culture has no control of their population growth such as family planning and does not have the infrastructure to support the nutritional needs then the death rate will increase causing their population to fall.
During the 20th century the world population grew at an unprecedented pace. This was mainly due to the advancement in crop production plus the better understanding of how certain food affect the human body. The better someone eats the better chance they have of living longer. Well-nourished mothers are more likely to produce health babies and help to ensure the health of the mother. There are many form of nutrition, certain food are better for you than others vitamins are another good source of nutrition. While many countries have benefited from all these nutritional advancements we still have countries that are see a reduction in their population because of the lack of food needed to maintain their current population.
While the world’s population has continues to grow we have a limited environment and we must ensure that it is able to supply the world’s food, shelter, water and energy for all our population now and in the future. We have the ability to measure the impact our growth in population is having on our environment and assess the global resources needed to support that population in the future. It is proven that if we continue to grow our population at the rate we are going and do not make changes in the way we are treating our environment we will destroy the world we live in. While all countries use the earth’s natural resources to live the larger developed countries do the most damage to our environment. When we start to see developing nation get richer we will see a much bigger impact to the environment that is already at its maximum capacity. As our population continues to grow we must consider the carrying capacity. Historically human population control was used to increase the world population by improving the conditions in which we live. The exception is China where due to their population issue they have implemented a one child per household policy. The world’s population has grown to the point that we must take action if we expect to survive.
We have review the population growth over the life of the earth and discuss many factors that had a positive and negative impact on that growth. Factors such as advancement in technology, the discovery and advancement in medicine and medical procedures, along with cultural and nutritional factors that have contributed to lengthening of lives. We also noted the impact the population growth has had on our environment and what need to be done to control our population in the future so we can ensure that we do not destroy our natural resources need to survive. As we continue to address the environmental issues we are currently encountering we must keep in mind not only how our current population is contributing to the problem but try and find a solution that will also address our future population. This is a global problem and we must address it as such. The United Nations must work to establish environmental rules that all countries must follow.
Rich. C, (2011): The Technological Influence on Population Growth Trends Retrieved from: http://rcantrell38.blogspot.com/2011/12/technological-influence-on-population.html
Gee. E, (ND): Population Growth Retrieved from http://www.deathreference.com/Nu-Pu/Population-Growth.html
Author Unknown, (ND): Human Impact on the Environment